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Title: The Chemistry of Plant Life

Author: Roscoe Wilfred Thatcher

Release date: August 9, 2010 [eBook #33394]
Most recently updated: January 6, 2021

Language: English

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Transcriber's Note

The original spelling and minor inconsistencies in the formatting have been
maintained. Obvious misprints were corrected and marked-up. The original text
will be displayed as a mouse-over pop-up.

The following words have been variably hyphenated in the original:
oxy(-)cumarin, tri(-)saccharides, sugar(-)like, mono(-)saccharides, sea(-)weeds,
di(-)sodium, foam(-)like, di(-)basic, aldo(-)hexoses, chromo(-)proteins,
galacto(-)octose, gluco(-)octose, keto(-)hexoses, ligno(-)celluloses,
manno(-)octose, para(-)pectic, di(-)saccharides, poly(-)saccharides. The variable
hyphenation has been retained in this version.

AGRICULTURAL AND
BIOLOGICAL PUBLICATIONS
Charles V. Piper, Consulting Editor

THE CHEMISTRY
OF PLANT LIFE

THE CHEMISTRY
OF PLANT LIFE

BY

ROSCOE W. THATCHER, M.A.,
D.Agr.
Dean of the Department of
Agriculture
and Director of the Agricultural
Experiment Stations.
University of Minnesota
(formerly Professor of Plant
Chemistry. University of
Minnesota)
First Edition
Second Impression

McGRAW-HILL BOOK COMPANY, Inc.
NEW YORK: 370 SEVENTH AVENUE

LONDON: 6 & 8 BOUVERIE ST., E. C. 4
1921

Copyright 1921, BY THE
McGRAW-HILL BOOK COMPANY, Inc.

Preface
The author has had in mind a two-fold purpose in the preparation of this
book. First, it is hoped that it may serve as a text or reference book for
collegiate students of plant science who are seeking a proper foundation upon
which to build a scientific knowledge of how plants grow. The late Dr. Charles
E. Bessey, to whom I owe the beginning of my interest in plant life, once said
to me: "The trouble with our present knowledge of plant science is that we
have had very few chemists who knew any botany, and no botanists who knew
any chemistry." This may have been a slightly exaggerated statement, even
when it was made, several years ago. But it indicated a very clear recognition
by this eminent student of plants of the need for a better knowledge of the
chemistry of plant cell activities as a proper foundation for a satisfactory
knowledge of the course and results of plant protoplasmic activities. It is
hoped that the present work may contribute something toward this desired end.

Second, the purpose of the writer will not have been fully accomplished
unless the book shall serve also as a stimulus to further study in a fascinating
field. Even the most casual perusal of many of its chapters cannot fail to make
clear how incomplete is our present knowledge of the chemical changes by
which the plant cell performs many of the processes which result in the
production of so many substances which are vital to the comfort and pleasure
of human life. Studies of the chemistry of animal life have resulted in many
discoveries of utmost importance to human life and health. It requires no great
stretch of the imagination to conceive that similar studies of plant life might
result in similar or even greater benefit to human life, or society, since it is

upon the results of plant growth that we are dependent for most of our food,
clothing, and fuel, as well as for many of the luxuries of life.

The material presented in the book has been developed from a series of
lecture-notes which was used in connection with a course in "Phyto-
chemistry" which was offered for several years to the students of the Plant
Science Group of the University of Minnesota. In the preparation of these
notes, extensive use was made of the material presented in such general
reference works as Abderhalden's "Biochemische Handlexikon" and
"Handbuch der Biochemischen Arbeitsmethoden," Oppenheimer's "Handbuch
der Biochemie des Menschen und der Tiere," Czapek's "Biochemie der
Pflanzen," Rohmann's "Biochemie," Frankel's "Descriptive Biochemie," and
"Dynamische Biochemie," Euler's "Pflanzenchemie," and Haas and Hill's
"Chemistry of Plant Products"; as well as of the most excellent series of
"Monographs on Biochemistry," edited by Plimmer, several numbers of which
appeared in print prior to and during the period covered by the preparation of
these lectures. Frequent use was made also of the many special treatises on
individual groups of compounds which are mentioned in the lists of references
appended to each chapter, as well as of articles which appeared from time to
time in various scientific journals.

Hence, no claim is made of originality for the statements presented herein,
except in an insignificant number of studies of enzyme action, and of the
possible physiological functions of certain specific compounds. The only
contributions which the writer has felt qualified to make to this general subject
are those of an intense personal interest in the chemistry of plant processes and
a viewpoint with reference to the relation of chemical processes to vital
phenomena which will be apparent as the various subjects are presented.

The text has been prepared upon the assumption that the students who will
use it will have had some previous training in elementary inorganic and
organic chemistry. A systematic laboratory course in organic preparations,

such as is required of students who are preparing to become professional
chemists, is not at all a necessary requisite to the understanding of the
chemistry of the different groups of plant compounds as here presented; but it
is assumed that the student will have had such previous training as is now
commonly given in a one-year collegiate course in "General Chemistry," or a
year's work in general inorganic chemistry followed by a brief course in
"Types of Carbon Compounds" or "Elements of Organic Chemistry," such as is
usually required of students who are preparing for advanced work in
agricultural science, in animal or human nutrition, etc.

An attempt has been made to arrange the material in such a way as to
proceed from simpler chemical principles and substances to those of more
complex structures. This results in an arrangement of the groups to be studied
in an order which is quite different than their biological significance might
suggest. It is believed, however, that in the end a more systematic
understanding and a more orderly procedure is obtained in this way than
would result from the treatment of the groups in the order of their relative
biological importance.

CONTENTS
Introduction Page
Development of biological science; characteristics
of protoplasm; plant and animal life, similarities and
differences; protoplasmic activity essentially
chemical changes; objects of study of the chemistry
of plant life xiii-xvi
CHAPTER I—Plant Nutrients
Definitions; the plant food elements; available and
unavailable forms; the value of the different soil
elements as plant foods; functions of the different
plant food elements in plant growth; inorganic plant
toxins and stimulants; references 1-15
CHAPTER II—Organic Components of Plants
Plants as synthetic agents; types of changes involved
in plant growth; groups of organic compounds found
in plants; physiological use and biological
significance defined; physiological uses of organic
groups 16-20
CHAPTER III—Photosynthesis
Definitions; physiological steps in photosynthesis;
formaldehyde, the simplest carbohydrate structure;
the condensation of formaldehyde into sugars;
theories concerning photosynthesis; the production
of starches and sugars; references 21-29
CHAPTER IV—Carbohydrates
Importance, nomenclature, and classification; 30-66
groups of carbohydrates; isomeric forms of
monosaccharides; chemical constitution of

monosaccharides; characteristic reactions of
hexoses; the occurrence and properties of
monosaccharides; disaccharides; trisaccharides;
tetrasaccharides; the relation of molecular
configuration to biochemical properties;
polysaccharides, dextrosans, levulosans, mannosans,
and galactosans; physiological uses and biological
significance of carbohydrates; references
CHAPTER V—Gums, Pectins, and Celluloses
Relation to carbohydrates; groups; the natural gums
and pentosans; mucilages; pectins; celluloses;
physiological uses of celluloses; referencess 67-75
CHAPTER VI—Glucosides
Definition; general structure; hydrolysis of the
natural glucosides; general properties; the phenol
glucosides; the alcohol glucosides; the aldehyde
glucosides; the oxycumarin glucosides; the
cyanophoric glucosides; the mustard-oil glucosides;
the pigment glucosides; the digitalis glucosides; the
saponins; physiological uses; biological
significance; references 76-93
CHAPTER VII—Tannins
General properties; occurrence; chemical
constitution; classes; some common tannins;
physiological uses; biological significance of
tannins in fruits; references 94-101
CHAPTER VIII—Pigments
Types and classes; the chlorophylls, chemical
constitution, similarity of chlorophyll and
hæmoglobin, properties of the chlorophylls; the
carotinoids, carotin, xanthophyll, lycopersicin, and
fucoxanthin; phycoerythrin and phycophæin; the
anthocyans; the anthoxanthins; the production of
ornamental pigments in flowers, etc.; the functions
of pigments; references 102-123
CHAPTER IX—Organic Acids, Acid Salts, and
Esters

Chemical constitution; some common organic acids;
physiological uses of organic acids; biological
significance of fruit acids and esters 124-128
CHAPTER X—Fats and Oils, Waxes, and Lipoids
General composition; fats and oils, occurrence,
chemical constitution, acids which occur in natural
fats, alcohols which occur in natural fats, hydrolysis
and synthesis of fats, extraction of oils from plant
tissues, identification of fats and oils, physiological
use; the waxes; the lipoids, lecithin, other plant
phosphatides, plant cerebrosides, physiological uses
of lipoids; references 129-145
CHAPTER XI—Essential Oils and Resins
Definitions, classes, occurrence; the essential oils;
the resins; physiological uses and biological
significance of essential oils; references 146-150
CHAPTER XII—The Vegetable Bases
Composition and groups; the plant amines;
alkaloids; the purine bases; the pyrimidines; the
nucleic acids, composition and uses; references 151-163
CHAPTER XIII—Proteins
Importance; general composition; amino-acids and
peptid units; individual amino-acids; composition of
the plant proteins; general properties of proteins;
classification; differences between plant and animal
proteins; extraction of proteins from plant tissues;
synthesis in plants; physiological uses; references 164-180
CHAPTER XIV—Enzymes
Reaction velocities; enzymes as catalysts; general
properties; extracellular and intracellular enzymes;
chemical nature; nomenclature and classification;
occurrence and preparation; general and individual
enzymes; nature of enzyme action; accelerators and
inhibitors; coenzymes and antienzymes; zymogens;
physiological uses; further studies needed;
references 181-201
CHAPTER XV—The Colloidal Condition

"Colloids" and "crystalloids"; the colloidal condition
a dispersion phenomenon; nomenclature and
classification; conditions necessary to the formation
of sols; gel-formation; general properties of
colloidal solutions; suspensoids and emulsoids;
adsorption; catalysis affected by the colloidal
condition; industrial applications of colloidal
phenomena; natural colloidal phenomena;
references 202-220
CHAPTER XVI—The Physical Chemistry of
Protoplasm
Heterogeneous structure of protoplasm; protoplasm
a colloidal gel; water; salts; osmotic pressure;
surface boundary phenomena; electrical phenomena;
acidity and alkalinity; summary; vital phenomena as
chemical and physical changes; references 221-238
CHAPTER XVII—Hormones, Auximones,
Vitamines, and Toxins
External and internal stimulants; hormones;
vitamines; auximones, toxins 221-238
CHAPTER XVIII—Adaptations
General discussion; adaptations, accommodations,
and adjustments; chromatic adaptations;
morphological adaptations; accommodations;
concluding statements 249-258
Index 259-268

INTRODUCTION
The history of biological science shows that the conceptions which men
have held concerning the nature of plant and animal growth have undergone a
series of revolutionary changes as the technique of, and facilities for, scientific
study have developed and improved. For a long time, it was thought that life
processes were essentially different in character than those which take place in
inanimate matter, and that the physical sciences had nothing to do with living
changes. Then, too, earlier students had only vague notions of the actual
structure of a living organism. Beginning with the earliest idea that a plant or
an animal exists as a unit organism, to be studied as such, biological science
progressed, first to the recognition and study of the individual organs which
are contained within the organism; then to the tissues which make up these
organs; then (with the coming into use of the microscope as an aid to these
investigations) to the cells of which the tissues are composed; then to the
protoplasm which constitutes the cell contents; and finally to the doctrine of
organic evolution as the explanation of the genealogy of plants and animals,
and the study of the relation of the principles of the physical sciences to the
evolutionary process. The ultimate material into which organisms are resolved
by this process of biological analysis is the cell protoplasm. But protoplasm is
itself made up of a complex system of definite chemical compounds, which
react and interact according to the laws of physical science. Hence, any study
of the chemistry of plant growth is essentially a study of the chemical and
physical changes which take place in the cell protoplasm.

Protoplasm differs from non-living matter in three respects. These are (1) its
chemical composition; (2) its power of waste and repair and of growth; and (3)
its reproductive power. From the standpoint of chemical composition,
protoplasm is the most complex material in the universe. It not only contains a
greater variety of chemical elements, united into molecules of enormous size
and complexity, but also a greater variety of definite chemical compounds than
exist in any other known mixture, either mineral or organic in type. One of the
first problems in the study of protoplasm is, therefore, to bring this great
variety of complex compounds into some orderly classification and to become
familiar with their compositions and properties. Again, living matter is
continually undergoing a process of breaking down as a result of its energetic
activities and of simultaneously making good this loss by the manufacture of
new protoplasm out of simple food materials. It also has the power of growth
by the production of surplus protoplasm which fills new cells, which in turn
produce new tissues and so increase the size and weight of individual organs
and of the organism as a whole. Hence, a second field of study includes the
chemical changes whereby new protoplasm and new tissue-building material
are elaborated. Finally, living material not only repairs its own waste and
produces new material of like character to it, but it also produces new masses
of living matter, which when detached from the parent mass, eventually begin
a separate existence and growth. Furthermore, the plant organism has acquired,
by the process of evolution, the ability not only to produce an embryo for a
successive generation but also to store up, in the tissues adjacent to it, reserve
food material for the use of the young seedling until it shall have developed
the ability to absorb and make use of its own external sources of food material.
So that, finally, every study of plant chemistry must take into consideration the
stored food material and the germinative process whereby this becomes
available to the new organism of the next generation. Also, the chemistry of
fertilization of the ovum, so that a new embryo will be produced, and the other
stimuli which serve to induce the growth phenomena, must be brought under
observation and study.

A further step in the development of biological science has been to separate
the study of living things into the two sciences of botany and zoology. From
the standpoint of the chemistry of the processes involved this segregation is
unfortunate. It has resulted in the devotion of most of the study which has been
given to life processes and living things to animal chemistry, or "physiological
chemistry." As a consequence, biochemistry, which deals with the living
processes of both plants and animals, is yet in its infancy; while
phytochemistry is almost a new science, yet its relation to the study of plants
can scarcely be less vital than is that of physiological chemistry to studies of
animal life.

The common conception that plant life and animal life are antithetical or
complementary to each other has much to justify it. Animals breathe in oxygen
and exhale carbon dioxide; while plants use the carbon dioxide of the air as a
part of the raw material for photosynthesis and exhale oxygen. Plants absorb
simple gases and mineral compounds as raw food materials and build these up
into complex carbohydrates, proteins, fats, etc.; while animals use these
complex compounds of plant origin as food, transforming parts of them into
various other forms of structural material, but in the end breaking them down
again into the simple gases and mineral compounds, which are expelled from
the body through the excretory organs. Thus it would seem that the study of
the chemistry of plant life and of animal life must necessarily deal with
opposite types of phenomena.

But one cannot advance far into the study of the biochemistry of plants and
animals before he discovers marked similarities in the chemical principles
involved. Many of the compounds are identical in structure, undergo similar
changes, and are acted upon by similar catalysts. Plant cells exhibit respiratory
activities, using oxygen and giving off carbon dioxide, in exactly the same
way that animal organisms do. The constructive photosynthetic processes of
green plants are regulated and controlled by a pigment, chlorophyll, which is
almost identical with the blood pigment, hæmatin", which regulates the vital

activities in the animal organism, differing from the latter only in the mineral
element which links the characteristic structural units together in the molecule.
Many other points of similarity in the chemistry of the life processes of plants
and animals will become apparent as the study progresses. It is sufficient now
to call attention to the fact that these vital processes, in either plants or
animals, are essentially chemical in character, and subject to study by the usual
methods of biochemical investigations.

The protoplasm of the cell is the laboratory in which all the changes which
constitute the vital activities of the plant take place. All of the processes which
constitute these activities—assimilation, translocation, metabolism, and
respiration—involve definite chemical changes. In so far as it is possible to
study each of these activities independently of the others, they have been
found to obey the ordinary laws of chemical reactions. Thus, the effect of the
variations in intensity of light upon photosynthesis causes increase in the rate
of this activity which may be represented by the ordinary responses of reaction
velocities to external stimuli. Similarly, the effect of rises in temperature upon
the rate of assimilation and upon respiration are precisely the same as their
effect upon the velocity of any ordinary chemical reaction. Within certain
definite ranges of temperature, the same statement holds true with reference to
the rate of growth of the plant, although the range of temperature within which
protoplasm lives and maintains its delicate adjustment to the four vital
processes of life is limited; beyond a certain point, further rise in temperature
does not produce more growth but rather throws the protoplasmic adjustment
out of balance and growth either slows up markedly or stops altogether.

Hence, we may say that the methods by which the plant machine
(protoplasm) accomplishes its results are essentially and definitely chemical in
character and may be studied purely from the standpoint of chemical reactions,
but the maintenance of the machine itself in proper working order is a vital
phenomenon which is largely dependent upon the external environmental
conditions under which the plant exists. A study of the phenomena resulting

from the colloidal condition of matter is throwing a flood of light upon the
mechanism by which protoplasm accomplishes its control of vital activities.
But we are, as yet, a long way from a complete understanding of how colloidal
protoplasm acquires and maintains its unique ability of self-regulation of the
conditions necessary to preserve its colloidal properties and of how it
elaborates the enzymes which control the velocity of the chemical reactions
which take place within the protoplasm itself and which constitute the various
processes of vital activity.

The object of this study of the chemistry of plant growth is to acquire a
knowledge of the constitution of the compounds involved and of the
conditions under which they will undergo the chemical changes which, taken
all together, constitute the vital processes of cell protoplasm.

CHEMISTRY OF PLANT LIFE

CHAPTER I

PLANT NUTRIENTS

There is some confusion in the use of the terms "nutrient," "plant food," etc.,
as applied to the nutrition and growth of plants. Strictly speaking, these terms
ought probably to be limited in their application to the organized compounds
within the plant which it uses as sources of energy and of metabolizable
material for the development of new cells and organs during its growth.
Botanists quite commonly use the terms in this way. But students of the
problems involved in the relation of soil elements to the growth of plants,
including such practical questions as are involved in the maintenance of soil
productivity and the use of commercial fertilizers for the growing of economic
plants, or crops, are accustomed to use the terms "plant foods," or "mineral
nutrients," to designate the chemical elements and simple gaseous compounds
which are supplied to the plant as the raw material from which its food and
tissue-building materials are synthetized. Common usage limits these terms to
the soil elements; but there is no logical reason for segregating the raw
materials derived from the soil from those derived from the atmosphere.

The essential difference between these raw materials for plant syntheses and
the organic compounds which are produced within the plants and used by
them, and by animals, as food, is that the former are inorganic and can furnish
only materials but no energy to the organism; while the latter are organic and

supply both materials and potential energy. It would probably be the best
practice to confine the use of the word "food" to materials of the latter type,
and several attempts have been made to limit its use in this way and to apply
some such term as "intake" to the simple raw materials which are taken into
the organism and utilized by it in its synthetic processes. But the custom of
using the words "food," or "nutrient," to represent anything that is taken into
the organism and in any way utilized by it for its nourishment has been
followed so long and the newer terms are themselves so subject to criticism
that they have not yet generally supplanted the loosely used word "food."

If such use is permitted, however, it is necessary to recognize that only the
green parts of green plants can use this inorganic "food," and that the colorless
plants must have organic food.

To avoid this confusion, the suggestion has recently been made that all of
the intake of plants and animals shall be considered as food, but that those
forms which supply both materials and potential energy to the organism shall
be designated as synergic foods, while those which contain no potential energy
shall be known as anergic foods. On this basis, practically all of the food of
animals, excepting the mineral salts and water, and all of the organic
compounds which are synthetized by plants and later used by them for further
metabolic changes, are synergic foods; while practically all of the intake of
green plants is anergic food.

It is with the latter type of food materials that this chapter is to deal; while
the following and all subsequent chapters deal with the organic compounds
which are synthetized by plants and contain potential energy and are,
therefore, capable of use as synergic food by either the plants themselves or by
animals. It will be understood, therefore, that in this chapter the word "food" is
used to mean the anergic food materials which are taken into and used by
green plants as the raw materials for the synthesis of organic compounds, with
the aid of solar energy, or that of previously produced synergic foods. In all

later chapters, the term "food" will be used to mean the organic compounds
which serve as the synergic food for the green parts of green plants and as the
sole supply of nutrient material for the colorless parts of green plants and for
parasitic or saprophytic forms (see page 16).

PLANT FOOD ELEMENTS

The raw materials from which the food and tissue-building compounds of
plants are synthetized include carbon dioxide, oxygen, water, nitrogen,
phosphorus, sulfur, potassium, calcium, magnesium, and iron. The two gases
first mentioned are derived directly from the air, through the respiratory organs
of the plant. Water is taken into the plant chiefly from the soil, through its
fibrous roots. All the other elements in the list are taken from the soil, nitrogen
being derived from decaying organic matter (the original source of the
nitrogen is, however, the atmosphere, from which the initial supply of nitrogen
is obtained by direct assimilation by certain bacteria and perhaps other low
forms of plant life), and the remaining ones from the mineral compounds of
the soil.

Carbon dioxide and oxygen, being derived from the air, are always available
to the leaves and stems of growing plants in unlimited supply; but the supply
available to a seed when germinating in the soil, or to the roots of a growing
farm crop, may sometimes become inadequate, especially in soils of a very
compact texture, or "water-logged" soils. In such cases, the deficiency of these
gaseous food elements may become a limiting factor in plant growth.

Water is often a limiting factor in plant growth. Experiments which have
been repeated many times and under widely varying conditions show that
when water is supplied to a plant in varying amounts, by increasing the
percentage of water in the soil in which the plant is growing by regular
increments up to the saturation point, the growth of the plant, or yield of the

crop, increases up to a certain point and then falls off because the excess of
water reduces the supply of air which is available to the plant roots. Hence,
abundance of water is, in general, a most essential factor in plant growth.

Under normal conditions of air and moisture supply, however, the plant food
elements which may be considered to be the limiting factors in the nutrition
and growth of plants are the chemical elements mentioned in the list above.

AVAILABLE AND UNAVAILABLE FORMS

The plant food materials which are taken from the soil by a growing plant
must enter it by osmosis through the semi-permeable membranes which
constitute the epidermis of the root-hairs, and circulate through the plant either
carried in solution in the sap or by osmosis from cell to cell. Hence, they must
be in water-soluble form before they can be utilized by plants. Obviously,
therefore, only those compounds of these elements in the soil which are
soluble in the soil water are available as plant food. The greater proportion of
the soil elements are present there in the form of compounds which are so
slightly soluble in water as to be unavailable to plants. The processes by which
these practically insoluble compounds become gradually changed into soluble
forms are chiefly the "weathering" action of air and water (particularly if the
latter contains carbonic acid) and the action of the organic acids resulting from
decaying animal or vegetable matter or secreted by living plants.

THE VALUE OF THE SOIL ELEMENTS AS PLANT
FOOD

Analyses of the tissues of plants show that they contain all of the elements
that are to be found in the soil on which they grew. Any of these elements

which are present in the soil in soluble form are carried into the plants with the
soil water in which they are dissolved, whether they are needed by the plant
for its nutrition or not. But in the case of those elements which are not taken
out of the sap to be used by the plant cells in their activities, the total amount
taken from the soil is much less than is that of the elements which are used in
the synthetic processes of the plant. Hence, much larger proportions of some
elements than of others are taken from the soil by plants. The proportions of
the different elements which are used by plants as raw materials for the
manufacture of the products needed for their growth varies with the different
species; but a certain amount of each of the so-called "essential elements" (see
below) is necessary to every plant, because each such element has a definite
rôle which it performs in the plant's growth. A plant cannot grow to maturity
unless a sufficient supply of each essential element comes to it from the soil.

From the standpoint of their relative value as raw materials for plant food,
the elements which are present in the soil may be divided into three classes;
namely, the non-essential, the essential and abundant, and the critical
elements.

The first class includes silicon, aluminium, sodium, manganese, and certain
other rarer elements which sometimes are found in soils of some special type,
or unusual origin. These elements seem to have no rôle to play in the nutrition
of plants; although silicon is always present in plant ash and sodium salts are
found in small quantities in all parts of practically all plants. Nearly all species
of plants can be grown to full maturity in the entire absence of these elements
from their culture medium. Occasional exceptions to this statement in the case
of special types of plants are known, and are of interest in special studies of
plant adaptations, but need not be considered here.

The second group includes iron, calcium, magnesium, and, generally, sulfur.
All of these elements are essential for plant growth, but are usually present in
the soil in ample quantities to insure a sufficient supply in available form for

all plant needs. Recent investigations have shown, however, that there are
many soils in which sulfur is present in such limited quantities that many
agricultural crops, when grown on these soils, respond favorably to the
application of sulfur-containing fertilizers. In such cases, sulfur is a "critical"
element.

The "critical" elements are those which are essential to the growth of all
plants and which are present in most soils in relatively small proportions and
any one may, therefore, be the limiting factor in plant growth so far as plant
food is concerned. These are nitrogen, phosphorus, potassium, and (possibly)
sulfur.

RÔLE OF PLANT FOOD ELEMENTS IN PLANT
GROWTH

The use which a plant makes of the elements which come to it from the soil
has been studied with great persistency and care by many plant physiologists
and chemists. Many of the reactions which take place in a plant cell are
extremely complicated, and the relation of the different chemical elements to
these is not easily ascertained. It is probable that the same element may play a
somewhat different rôle in different species of plants, in different organs of the
same plant, or at different stages of the plant's development. But the usual and
most important offices of each element are now fairly well understood, and are
briefly summarized in the following paragraphs. It should be understood that a
thorough and detailed discussion of these matters, such as would be included
in an advanced study of plant nutrition, would reveal other functions than
those which are presented here and would require a more careful and more
exact method of statement than is suitable here. However, the general
principles of the utilization of soil elements by plants for their nutrition and
growth may be fairly well understood from the following statements.

Nitrogen is a constituent of all proteins (see Chapter XIII). Proteins are
apparently the active chemical components of protoplasm. Since it is in the
protoplasm of the green portions, usually foliage, of plants that the
photosynthesis of carbohydrates and the synthesis of most, or all, of the other
tissue-building materials and reserve food substances of the plant takes place,
the importance of nitrogen as a plant food can hardly be over-emphasized.
Nitrogen starvation produces marked changes in the growth of a plant. Leaves
are stunted in growth and a marked yellowing of the entire foliage takes place;
in fact, the whole plant takes on a stunted or starved appearance. Abundance of
nitrogen, on the other hand, produces a rank growth of foliage of a deep rich
color and a luxuriant development of tissue, and retards the ripening process.
In the early stages of growth, the nitrogen is present most largely in the leaves;
but when the seeds develop, rapid translocation of protein material into the
seeds takes place, until finally a large proportion of the total supply is
deposited in them.

Nitrates are the normal form of nitrogen in the soil which is available to
plants. During germination and early growth, the young seedling uses amino-
acids, etc., derived from the proteins stored in the seed, as its source of
nitrogen; and experiments have shown that similar forms of soluble organic
nitrogen compounds can be successfully fed to the seedling as an external food
supply. Soluble ammonium salts can be utilized as sources of nitrogen by most
plants during later periods of growth, particularly by the legumes. But for
most, if not all, of the common farm crops whose possibilities in these respects
have been studied, it has been found that a unit of nitrogen taken up as a nitrate
is very much more effective in promoting growth, etc., than is the same unit of
nitrogen in the form of ammonium salts.

While the proteins are finally stored up largely in the seeds, or other storage
organs, they are actively at work during the growing period in the cells of the
foliage parts of the plant. Hence, the popular statement that "nitrogen makes
foliage" is a fairly accurate expression of its rôle. Inordinate production of

straw in cereal crops and of leaves in root crops often results from liberal
supplies of available nitrogen in the soil early in the growing season. If the
crops develop to normal maturity, this excessive foliage growth has no harmful
results, as the surplus material which has been elaborated is properly
translocated into the desired storage organs; but, unfortunately, the retarding
effect of the surplus nitrogen supply upon the date of maturing of the crop is
often associated with premature ripening of the plants from other causes, with
the consequence that too large a proportion of the valuable food material is left
in the refuse foliage material of the crop. Crops which are grown solely for
their leaves, such as hay crops, lettuce, cabbage, etc., profit greatly by
abundant supplies of available nitrogen; although when foliage growth is
stimulated in this way the tissue is likely to be thin-walled and soft rather than
firm and solid.

Phosphorus is likewise an extremely important element in plant nutrition.
But phosphorus starvation produces no such striking visible effects upon the
growth of the plant as does lack of nitrogen. Abundance of available
phosphorus early in the plant's life greatly stimulates root growth, and later on
it undoubtedly hastens the ripening process; hence, this element seems to act
as the exact antithesis of nitrogen.

The rôle of phosphorus, or of phosphates, in the physiological processes of
the cell seems to be difficult to discover. The element itself is a constituent of
some protein complexes and of the lecithin-like bodies (see page 141) which
are supposed by some investigators to play an important part in determining
the rate of chemical changes which take place in the cell and the movement of
materials into and out of it. It is an essential constituent of the nucleus, and a
meager supply of phosphorus retards, or inhibits, mitotic cell-division.
Photosynthesis of sugars and the condensing of these into starch or cellulose
takes place in plants in the absence of available phosphorus; but the change of
these insoluble carbohydrates back again into soluble and available sugar
foods does not.

Phosphorus is taken from the soil by plants in the form of phosphates. Much
study has been given to the problem of the proper supply of available soil
phosphates for economic crop production. Any discussion of soil fertility and
fertilization which did not devote large attention to the conditions under which
phosphates become available as plant food would be wholly inadequate; but
such a discussion would be out of place here.

The final result of an ample supply of phosphates in hastening the ripening
process and stimulating seed production, as contrasted with that of an over-
supply of nitrogen, has led to the popular statement that "phosphates make
seeds." This statement, while not strictly accurate, is a fairly good summary of
the combined results of the rôle of phosphorus in the plant economy. Large
amounts of phosphorus are stored in the seeds. The two facts that large
amounts of these compounds are thus available to the young seedling and that
relatively large proportions of phosphates are taken from the soil by the plant
during its early stages of growth are undoubtedly connected with the need for
rapid cell-division at these periods in the plant's life.

Potassium.—The popular expression that "potash makes sugars and starch"
is a surprisingly accurate description of the rôle of this element in plant
metabolism. Either the photosynthesis of starch, or the changes necessary to its
translocation (it is not yet certain which) is so dependent upon the presence of
potassium in the cell sap that the whole process stops at once if an insufficient
supply is present. The production and storage of sugar, or starch, in such root
crops as beets, potatoes, etc., diminishes in direct proportion with a decreasing
supply of potassium as plant food. The grains of the cereal crops become
shrunken as a result of potassium starvation; and are plump and well filled
with starch in the endosperm when sufficient potassium is available for the
crop's needs.

The general tone and vigor of growth of the plant is largely dependent upon
an ample potassium supply; potash-hungry plants, like those which have been

weakened by any other unfavorable conditions, have been found to be more
susceptible to injury by disease, than those which are well nourished with this
food element. But potassium-starvation does not produce any pathological
condition of the cell contents; its absence simply prevents the possibility of the
development of the necessary carbohydrates for vigorous growth.

There is no known difference in the availability, or effectiveness, of
potassium from the different forms of compounds containing it which may be
present in the soil. Apparently, the only essential is that the compound shall be
soluble so that it can be absorbed into the plant through the root-hairs. Of
course, the acid radical to which the basic potassium ion is attached may, in
itself, have some beneficial or deleterious influence which gives to the
compound as a whole some important effect in one case, which might not
follow in the case of another type of compound; but the relative efficiency as
plant food of a given unit of potassium seems to be the same regardless of the
nature of the compound in which it is present.

Calcium is an essential plant food element but its physiological use has not
yet been definitely established. It seems to stimulate root-development, and
certainly gives vigor and tone to the whole plant. It is commonly believed that
calcium is in some way connected with the development of cell-wall material.
It has been reported that the stems of grasses and cereal plants become stiffer
in the presence of ample calcium, but this may be due to greater turgidity
rather than to strengthened cell-walls. Calcium remains in the leaves or stem as
the plant ripens, but it is not clear that this has anything to do with the stiffness
or weakness of the stem, or straw, of the plant. Experiments with algæ have
shown that in the absence of calcium salts mitotic cell division takes place,
showing that the nucleus functions properly, but the formation of the new
transverse cell-wall is retarded. This is the only direct evidence that has been
reported that calcium has any connection with cell-wall formation.

Certain species of plants, notably many legumes, require such large amounts
of calcium salts for their growth as to give to them the popular appellation of
"lime-loving plants." Other plants, known as "calciphiles," while not actually
showing abnormally large percentages of calcium in their ash, flourish best on
soils rich in lime. On the other hand, certain other species, known as
"calcifuges," will not grow on soils which are even moderately rich in lime; in
what respect these differ in their vital processes from others which demand
large amounts of calcium, or those which flourish on soils rich in lime, has not
been determined, however.

The beneficial effect of alkaline calcium compounds in the soil, in
correcting injurious acidity, in improving the texture of clay soils, and in
promoting the proper conditions for bacterial growth, is well known; but this
has no direct connection with the rôle of calcium as plant food. Furthermore,
calcium salts in the soil have a powerful influence in overcoming the harmful,
or toxic, effects of excessive amounts of soluble salts of magnesium, sodium,
or potassium, in the so-called "alkali soils" (i.e., those which contain excessive
amounts of water-soluble salts). The probable explanation for this fact is
pointed out in a later paragraph of this chapter (see page 14); but this property
of calcium probably has no connection with its physiological uses as plant
food.

Magnesium, like phosphorus, is finally stored up mostly in the seeds, not
remaining in the leaves and stems, as do calcium and potassium. This fact,
together with other evidence obtained from experiments in growing plants in
culture solutions containing varying amounts of this element, has led certain
investigators to the conclusion that the rôle of magnesium is to aid in the
transport of phosphorus, particularly from older to more rapidly growing parts
of the plant. More recent investigations have shown, however, that magnesium
has other roles which are probably more specific and more important than this
one. It is now known that magnesium is a definite constituent of the
chlorophyll molecule serving, as will be shown (see Chapter VIII), as the

means of linkage between its essential component organic groups. Because of
this fact, magnesium-starvation produces etiolated plants, which cannot
function normally. Further, magnesium seems to be necessary for the
formation of fats, apparently standing in a similar relation to fat-formation to
that of potassium to carbohydrate-formation. This view is supported by the
observations that when algæ are grown in magnesium-free solutions they
contain no fat globules and that oily seeds are richer in magnesium than are
those which store up starch as their reserve food material. Observers of the
second of these phenomena have failed to note, however, that oily seeds are
likewise richer in phosphorus than are starchy ones, and that the presence of
larger proportions of magnesium in such seeds may, perhaps, be related to
phosphorus-translocation rather than to fat-formation.

Whatever relation magnesium may have to fat-formation, or to the
translocation of phosphorus, it is evident that these are rôles quite apart from
its use as a constituent element in chlorophyll. As yet, no explanation of how it
aids in these other synthetic processes has been advanced.

On the other hand, an excess of soluble magnesium salts in the soil produces
definite toxic effects upon plants, magnesium compounds being known to be
among the most destructive of the "alkali soil" salts. Calcium salts are
remarkably efficient in overcoming these harmful effects of magnesium salts.
On this account, a large amount of experimental study has been given to the
question of the calcium-magnesium ratio in plants. Numerous analyses of
plant ashes have established the fact that there is a fairly definite ratio of this
kind, which ratio, however, varies with the species of plant and is not
correlated with the ratio of these elements present in the soil on which the
plant grows, as was formerly believed. Cereal plants, as a rule, contain
approximately twice as much lime as magnesia; while leafy plants (tobacco,
cabbage, etc.) usually contain about four times as much calcium oxide as
magnesium oxide.

Iron is essential to chlorophyll-formation. It is not a constituent of the
chlorophyll molecule, as is magnesium; but in the absence of iron from the
culture solution, a plant fails to produce chlorophyll and a green plant which is
deprived of a supply of iron rapidly becomes etiolated. The way in which iron
is related to chlorophyll-formation is not known.

Iron is taken from the soil by plants in the smallest proportions of any of the
essential elements. Only soluble ferric compounds seem to serve as a suitable
source of supply of the element; ferrous compounds being usually highly toxic
to plants.

Sulfur is an essential element of plant food. The amounts required by plants
were supposed, until recently, to be relatively small. This was due to the fact
that earlier studies took account only of the sulfur which, on analysis, appeared
as sulfates in the ash. Improved methods of analysis, which insure that the
sulfur which is present in the plant tissue in organic combinations is oxidized
under such conditions that it is not lost by volatilization during the combustion
of the material, have shown that the total sulfur which is present in many
plants approaches the quantity of phosphorus which is present in the same
tissue. Furthermore, recent field and pot experiments have shown that at least a
considerable part of the beneficial effects of many fertilizers, which has
previously been attributed to the calcium, potassium, or phosphorus which
they contain, is actually due to the sulfur present as sulfates in the fertilizers
used.

Sulfur occurs in the organic compounds of plants, associated with
phosphorus. It seems probable that its physiological uses are similar to those
of the latter element; but there is as yet no experimental evidence to establish
its exact rôle in the economy of plant growth. It appears to be needed in largest
proportion by plants which contain high percentages of nitrogen in their
foliage, such as the legumes. There is some evidence that sulfur has a
particular rôle in promoting the growth of bacteria, and it may be that the

percentages of total sulfur which are found in the tissues of legumes are due to
the presence of the symbiotic nitrogen-gathering bacteria in the nodules on the
roots of these plants. This point has not yet been investigated, however.

Sodium is probably not essential to plant growth, although it is present in
small proportions in the ash from practically all plants. In cases of insufficient
supply of potassium, sodium can apparently perform at least a part of the rôle
of the former element; but this seems not to be a normal relationship or use.

Chlorine is found in small amounts in the sap and in the ash of nearly all
plants. However, it does not appear to be essential to the growth of a plant,
except possibly in the case of certain species, such as asparagus, buckwheat,
and, perhaps, turnips and some other root crops. Whether the benefit which
these crops derive from the application of common salt to the soil in which
they are growing is due to the direct food value of either the chlorine, or the
sodium, or to some indirect effect, is not yet known. The presence of chlorine
in the sap of plants is undoubtedly due to the inevitable absorption of soluble
chlorides from the soil and apparently has no connection with the nutritional
needs of the plant.

Silicon is always considered as a non-essential element, although it occurs
in such large proportions in some plants as to indicate that it cannot be wholly
useless. It accumulates in the stems of plants, chiefly in the cell-wall, and has
sometimes been supposed to aid in giving stiffness to the stems. But large
numbers of analyses have failed to show any direct correlation between the
stiffness of straw of cereal plants and the percentage of silicon which they
contain. Further, plants will grow to full maturity and with erect stems when
no silicon is present in the mineral nutrients which are furnished to them. On
the other hand, certain experiments appear to indicate that silicon can perform
some of the functions of phosphorus, if soluble silicates are supplied to
phosphorus-starved plants. But under normal conditions of plant nutrition, it
seems to have no such function.

INORGANIC PLANT TOXINS AND STIMULANTS

Much study has been given during recent years to the question of the
supposed poisonous, or toxic, effects upon plants of various soil constituents.
There seems to be no doubt that certain organic compounds which are
injurious to plant life are often present in the soil, either as the normal
excretions of plant roots or as products of the decomposition of preceding
plant growths. A consideration of these supposedly toxic organic substances
would be out of place in this discussion of mineral soil nutrients. But there
seems to be no doubt that there may also be mineral substances in the soil
which may sometimes exert deleterious influences upon plant growth. In fact,
most metallic salts, except those of the few metals which are required for plant
nutrition, appear to be toxic to plants. The exact nature of the physiological
effects which are produced by these mineral toxins is not clearly understood;
indeed, it is probably different in the case of different metals. Further, it is
certain that both the stimulating and the toxic effect of metallic compounds
upon low forms of plants is quite different from the effects of the same
substances upon the more complex tissues of higher plants, a fact which is
utilized to advantage in the application of fungicides for the control of
parasitic growths on common farm crops.

Among the elements whose physiological effects upon higher plants, such
as the cereal crops, etc., when their soluble compounds are present in the soil,
have been carefully studied, there are three fairly distinct types of injurious
mineral elements. The first of these, represented by copper, zinc, and arsenic,
apparently exert their toxic effect regardless of the proportion in which they
are present in the nutrient solution which is presented to the plant; although the
degree of injury varies with the amount of injurious substance present, of
course. The second type, of which boron and manganese are representatives,
apparently exerts a definite stimulating effect upon plants when supplied to
them in concentrations below certain clearly defined limits; but are toxic in
concentrations above these. The third includes many soluble salts of

magnesium, sodium, potassium, etc., which while either innocuous or else
definite sources of essential plant foods when in lower concentrations, become
highly toxic, or corrosive, when present in the soil solution in concentrations
above the limits of "toleration" of individual plants for these soluble salts. The
tolerance shown by the different species of plants toward these soluble salts
(the so-called "alkali" in soils) varies widely; indeed, there seems to be
considerable variation in the resistance of different individual plants of the
same species to injury from this cause.

With reference to the toxic effect of the third type of substances, i.e., the
common soluble salts, it is known that single salts of potassium, magnesium,
sodium, or calcium, in certain concentrations, are toxic to plants, while
mixtures of the same salts in the same concentrations are not. Thus, solutions
of sodium chloride, magnesium sulfate, potassium chloride, and calcium
chloride which, when used singly, killed plants whose roots were immersed in
them for only a few minutes, formed when mixed together a nutrient solution
in which the same plants grew normally. The remarkable remedial effect of
calcium salts in overcoming the injurious effects of other soluble salts has
already been mentioned. One explanation of these relationships between
mineral soil constituents and the living plant is that the life phenomena depend
upon a balanced adjustment between the compounds of these different mineral
elements with the proteins (producing the so-called "metal proteids") which
constitute the active material of the cell protoplasm. According to this theory,
any excess or deficiency of any one or more of these elements in the plant
juices which surround a given cell will, of course, cause an interchange with
the mineral components of the supposed "metal proteids" which upsets the
assumed essential balance between them, with disastrous results. A more
recent, and much more satisfactory, explanation of the "antagonism" between
mineral elements in their toxic effects upon plants, which has both theoretical
and experimental confirmation, is that single salts disturb the colloidal
condition (see Chapter XV) of the protoplasm of the plant cells in such a way
as to destroy its permeability to nutrient substances, while mixtures of salts

restore the proper state of colloidal dispersion and permit the normal
functioning of the protoplasm.

It is apparent from the above brief discussions that the rôle of the different
soil elements as plant food, and their relations to the complex processes which
constitute plant growth, afford an interesting and promising field for further
study.

References

Brenchley, Winifred E.—"Inorganic Plant Poisons and
Stimulants," 106 pages, 18 figs., Cambridge, 1914.

Hall, A. D.—"Fertilizers and Manures," 384 pages, 7 plates,
London, 1909.

Hall, A. D.—"The Book of the Rothamsted Experiments," 294
pages, 49 figs., 8 plates, London, 1905.

Hopkins, C. G.—"Soil Fertility and Permanent Agriculture," 653
pages, Chicago, 1910.

Hilgard, E. W.—"Soils," 593 pages, 89 figs., New York, 1906.

Loew, O.—"The Physiological Rôle of Mineral Nutrients," U. S.
Department of Agriculture, Bureau of Plant Industry, Bulletin No.
45, 70 pages, Washington, D. C., 1903.

Russell, E. J.—"Soil Conditions and Plant Growth," 243 pages, 13
figs., Monographs on Biochemistry, London, 1917. (3d ed.)

Whitney, M.—"A Study of Crop Yields and Soil Composition in
Relation to Soil Productivity," U. S. Department of Agriculture,

Bureau of Soils, Bulletin No. 57, 127 pages, 24 figs., Washington,
D. C., 1909.

CHAPTER II

THE ORGANIC COMPONENTS OF PLANTS

From the standpoint of their ability to synthetize synergic foods (see page 2)
from inorganic raw materials, plants may be divided into two types; namely,
the autotrophic, or self-nourishing, plants, and the heterotrophic plants.

Strictly speaking, only those plants whose every cell contains chlorophyll
are entirely self-nourishing; and some parts, or organs, of almost any
autotrophic plant are dependent upon the active green cells of other parts of the
plant for their synergic food. Furthermore, if the term is used in a very wide
sense, green plants are more than self-nourishing, they really nourish all living
things. But the general significance of the term "autotrophic plants" is
apparent.

"Heterotrophic plants" must, of necessity, get food, either directly or
indirectly, from some other plant which can synthetize synergic foods or, in a
few cases, from animal organic matter. If they do this by feeding upon the
organic compounds of other living organisms, they are known as "parasites";
while if they secure their organic food from the tissues or debris of dead
organisms, they are called "saprophytes." The heterotrophic plants are chiefly
the bacteria and fungi; although a few seed-plants are devoid of chlorophyll or

have nutritive habits similar to those of the non-green plants, and a few species
are semi-parasitic or semi-saprophytic.

It is obvious that the metabolic processes of the autotrophic plants are very
different from those of the heterotrophic type of plants. These differences
constitute a most interesting field of study for plant physiologists. But the
nature of the chemical compounds themselves and of the chemical changes
involved in their transformations is not radically different in the two types of
plants, the essential difference being in the preponderance of one kind of
activities, or chemical reactions, over another in bringing about the metabolic
processes which are characteristic of each particular species. Hence, it does not
seem necessary, or desirable, in this study of the chemistry of plant growth, to
present as detailed a consideration of the differences in metabolic activity of
the different types of plants as complete accuracy of statement in all cases
might demand. We will, instead, discuss the organic chemical components of
plant tissues and the reactions which they undergo, using the more common
type of autotrophic plants as the illustrative material in most cases.

Hence, it will be understood that in all the following discussions of plant
activities, except where specific exceptions are definitely mentioned, it is the
green, or autotrophic, plants to which reference is made in each case.

From the standpoint of the sum total of its activities, a green plant is
essentially an absorber of solar energy and a synthetizer of organic substances.
Each individual autotrophic plant takes up certain amounts of the anergic
foods which are discussed in the preceding chapter and manufactures from
them a great variety of complex organic compounds, using the energy of the
sun's rays, absorbed by chlorophyll, as the source for the energy necessary to
accomplish these synthetic reactions. The ultimate object of these processes is
to produce seeds, each containing an embryo and a sufficient supply of food
for the young plant of the next generation to use until it has developed its own
synthetic organs; or (in the case of perennials) to store up reserve food

materials with which to start off new growth after a period of rest and often of
defoliation. To be sure, animals and men often interfere with the completion of
the life cycle of the plant, and utilize the seeds or stored food material for their
own nutrition, but this is a biological relation which has no influence upon the
nature of the plant's own activities.

Since all of these synthetic reactions must go on at ordinary temperatures,
active catalyzers are necessary. These the plant provides in the form of
enzymes (see Chapter XIV) which are always present in active plant
protoplasm. Proper conditions for rapid chemical action are further assured by
the colloidal nature (see Chapter XV) of the protoplasm itself.

TYPES OF CHEMICAL CHANGES INVOLVED IN
PLANT GROWTH

The whole cycle of chemical changes which is involved in plant growth
represents the net result of two opposite processes; the first of these is a
constructive one which has at least three different phases: namely, a synthesis
of complex organic compounds, the translocation of this synthetized material
to the centers of growth, and the building up of this food material into tissues
or reserve supplies; and the second is a destructive process of respiration
whereby carbohydrate material is broken down, potential energy is released,
and carbon dioxide is excreted.

The synthetic processes which take place in plants are of two types; namely,
photosynthesis, in which sugars are produced, and another, which has no
specific name, whereby proteins are elaborated. The translocation of the
synthetized material involves the change of insoluble compounds into soluble
ones, effected by the aid of enzymes. For storage purposes, the soluble forms
are usually, though not always, condensed again into more complex forms,

these latter changes requiring much less energy than do the original syntheses
from raw materials.

The destructive process, respiration, is characteristic of all living matter,
either plant or animal organisms. It takes place continuously throughout the
whole life of a plant. During rapid growth it is overshadowed by the results of
the synthetic process, but during the ripening period in which the seed is
matured, and during the germination of the seed itself, growth is practically at
a standstill and the respiratory, destructive action predominates, so that the
plant actually loses weight.

GROUPS OF ORGANIC COMPOUNDS FOUND IN
PLANTS

As a result of their various synthetic and metabolic activities, a great variety
of organic compounds is produced by plants. Certain types of these
compounds, such as the carbohydrates and proteins, are necessary to all plants
and are elaborated by all species of autotrophic plants. Other types of
compounds are produced by many, but not all, species of plants; while still
others are found in only a few species. It is fairly easy to classify all of these
compounds into a few, well-defined groups, based upon similarity of chemical
composition. These groups are known, respectively, as the carbohydrates and
their derivatives, the glucosides and tannins; the fats and waxes; the essential
oils and resins; organic acids and their salts; the proteins; the vegetable bases
and alkaloids; and the pigments. A consideration of these groups of
compounds, as they are synthetized by plants, constitutes the major portion of
the study of the chemistry of plant life as presented in this book. Following the
discussion of the compounds themselves, the chapters dealing with enzymes,
with the colloidal nature of protoplasm, and with the supposed accessory
stimulating agencies, aim to show how the manufacturing machine known as

the plant cell accomplishes its remarkable results, so far as the process is now
understood.

PHYSIOLOGICAL USES AND BIOLOGICAL
SIGNIFICANCE

In connection with the discussion of each of the above-mentioned groups of
organic components of plants, an attempt will be made to point out what
significance these particular compounds have in the plant's life and growth.
Certain terms will be used to designate different rôles, which it is probably
necessary to define.

There may be two possible explanations of, or reasons for, the presence of
any given type of compound in the tissues of any particular species of plant.
First, it may be supposed that this particular type of compounds is elaborated
by the plant to satisfy its own physiological needs, or for the purpose of
storing it up in the seeds as synergic food for the growth of the embryo, in
order to reproduce the species. For this rôle of the various organic food
materials, etc., we will employ the term "physiological use." On the other
hand, it is often conceivable that certain types of compounds, which have
properties that make them markedly attractive (or repellent) as a food for
animals and men, or which are strongly antiseptic in character, or which have
some other definite relationship to other living organisms, have had much to
do with the survival of the particular species which elaborates them, in the
competitive struggle for existence; or have been developed in the plant by the
evolutionary process of "natural selection." For this relation of the compound
to the plant's vital needs, we will use the term "biological significance." Such a
segregation of the rôles which the different compounds play in the plant's
economy may be more or less arbitrary in many cases; but it will be clear that
when physiological uses are discussed, reference is being made to the plant's

own internal needs; while the phrase biological significance will be
understood to refer to the relation of the plant to other living organisms.

PHYSIOLOGICAL USES OF THE ORGANIC
COMPONENT GROUPS

From the standpoint of the rôle which each plays in the plant economy, the
several groups of organic compounds may be roughly divided into three
classes. These are: (a) the framework materials, including gums, pectins, and
celluloses; (b) synergic foods, including carbohydrates, fats, and proteins; and
(c) the secretions, including the glucosides, volatile oils, alkaloids, pigments,
and enzymes.

The framework material, as the name indicates, constitutes the cell-wall and
other skeleton substances of the plant. It is made up of carbohydrate
complexes, produced by the cell protoplasm from the simpler carbohydrates.

The synergic foods, or "reserve foods" as they are sometimes called,
produced by the excess of synthetized material over that needed for the
immediate use of the plant, are accumulated either in the various storage
organs, to be available for future use by the plant itself or by its vegetative
offspring, or in the seed, to be available to the young seedling of the next
generation. Proteins not only serve as reserve food materials but also make up
the body of the living organism itself. Carbohydrates and fats serve as synergic
and reserve foods.

The secretions may be produced either in ordinary cells and found in their
vacuoles, or in special secretory cells and stored in cavities in the secreting
glands (as in the leaves of mints, skin of oranges, etc.), or in special ducts (as
in pines, milkweeds, etc.) or on the epidermis (as the "bloom" of plums,
cabbages, etc., the resinous coating of many leaves, etc.). As a general rule, the

glucosides, pigments, and enzymes are the products of unspecialized cells and
have some definite connection with the metabolic processes of the plant; while
the volatile oils and the alkaloids are usually secreted by special cells and have
no known rôle in metabolism.

CHAPTER III

PHOTOSYNTHESIS

Photosynthesis is the process whereby chlorophyll-containing plants, in the
presence of sunlight, synthetize organic compounds from water and carbon
dioxide. The end-product of photosynthesis is always a carbohydrate.
Chemical compounds belonging to other groups, mentioned in the preceding
chapter, are synthetized by plants from the carbohydrates and simple raw
materials; but in such cases the energy used is not solar energy and the process
is not photosynthesis.

Under the ordinary conditions of temperature, moisture supply, etc.,
necessary to plant growth, photosynthesis will take place if the three essential
factors, chlorophyll, light, and carbon dioxide are available.

PHYSIOLOGICAL STEPS IN PHOTOSYNTHESIS

There are five successive and mutually dependent steps in the process of
photosynthesis, as follows:

(1) There must be a gas exchange between the plant tissue and the
surrounding air, by means of which the carbon dioxide of the air may reach the

protoplasm of the chlorophyll-containing cells.

(2) Radiant energy must be absorbed, normally that of sunlight, although
photosynthesis can be brought about by the energy from certain forms of
artificial light.

(3) Carbon dioxide and water must be decomposed by the energy thus
absorbed, and the nascent gases thus produced combined into some synthetic
organic compound, with a resultant storage of potential energy.

(4) This first organic synthate must be condensed into some carbohydrate
suitable for translocation and storage as reserve food.

(5) The oxygen, which is a by-product from the decomposition of the water
and carbon dioxide and the resultant synthetic process, must be returned to the
air by a gas exchange.

Of the five steps in this process, the first two and the last are essentially
purely physical phenomena, the chemical changes involved being those of the
third and fourth steps. Hence, it is only these two parts of the process which
need be taken into account in a consideration of the chemistry of
photosynthesis.

FORMALDEHYDE, THE SIMPLEST
CARBOHYDRATE STRUCTURE

The simplest carbohydrates known to occur commonly in plant tissues are
the hexoses (see Chapter IV) having the formula C6H12O6, which is just six
times that of formaldehyde, CH2O. Also, it is known that formaldehyde easily,
and even spontaneously, polymerizes into more complex forms having the
general formula (CH2O)n; trioxymethylene, C3H6O3, being a well-known

example. Further, both trioxymethylene and formaldehyde itself can easily be
condensed into hexoses, by simple treatment with lime water as a catalytic
agent. Hence, it is commonly believed that formaldehyde is the first synthetic
product resulting from photosynthesis, that this is immediately condensed into
hexose sugars, and that these in turn are united into the more complex
carbohydrate groups which are commonly found in plants (see Chapter IV).

There is considerable experimental confirmation of the soundness of this
view. The whole photosynthetic process takes place in chlorophyll-containing
plant tissues with astonishing rapidity, sugars, and even starch, appearing in
the tissues almost immediately after their exposure to light in the presence of
carbon dioxide. Hence, any intermediate product, such as formaldehyde, is
present in the cell for only very brief periods and in very small amounts. But
small amounts of formaldehyde can often be detected in fresh green plant
tissues and, as will be pointed out below, the whole process of photosynthesis,
proceeding through formaldehyde as an intermediate product, can be
successfully duplicated in vitro in the laboratory.

Assuming, then, that formaldehyde is the first photosynthetic product in the
process of the production of carbohydrates from water and carbon dioxide, the
simple empirical equation for this transformation would be

H2O + CO2 = CH2O + O2.

It is apparent, however, that the process is not so simple as this hypothetical
reaction would indicate, as water and carbon dioxide can hardly be conceived
to react together in any such simple way as this. Various theories as to the
exact nature of the steps through which the chemical combinations proceed
have been advanced. A discussion of the experimental evidence upon which
these are based and of the conclusions which seem to be justified from these
experimental studies is presented below. The only value which may be
attached to the empirical equation just presented is that it does accurately
represent the facts that a volume of oxygen, equal to that of the carbon dioxide

consumed in the process, is liberated and that formaldehyde is the synthetical
product of the reactions involved.

It should be noted, in this connection, that formaldehyde is a powerful plant
poison and that few, if any, plant tissues can withstand the toxic effect of this
substance when it is present in any considerable concentration. Hence, it is
necessary to this whole conception of the relation of formaldehyde to the
photosynthetic process, to assume that, however rapidly the formaldehyde may
be produced in the cell, it is immediately converted into harmless carbohydrate
forms.

THE CONDENSATION OF FORMALDEHYDE
INTO SUGARS

As has been mentioned, it is easily possible to cause either formaldehyde, or
trioxymethylene, to condense into C6H12O6, using milk of lime as a catalyst.
Of course, no such condition as this prevails in the plant cell, and the
mechanics of the protoplasmic process may be altogether different from those
of the artificial syntheses. Furthermore, the hexose produced by the artificial
condensation of these simpler compounds is, in every case, a non-optically
active compound, while all natural sugars are optically active (see Chapter IV).
Emil Fischer has succeeded, however, by a long and round-about process
which need not be discussed in detail here, in converting the artificial hexose
into glucose and fructose, the optically-active sugars which occur naturally in
plant tissues. The condensation of formaldehyde directly into glucose and
fructose in the plant cell is brought about by some process the nature of which
is not yet understood. Probably synthetic enzymes (see Chapter XIV), whose
nature and action have not yet been discovered, come into play. It is a
noteworthy fact, however, that the mechanics of this apparently simple
chemical change, upon which the whole nutrition of the plant depends, and

which furnishes the whole animal kingdom, including the human race, with so
large a proportion of its food supplies, is as yet wholly unknown.

It is the common practice to represent the whole results of the
photosynthetic action by the empirical equation

6H2O + 6CO2 = C6H12O6 + 6O2;

but here again the only value to be attached to such an algebraic expression
is that it accurately represents the gaseous exchange of carbon dioxide and
oxygen involved in the process. Certainly, it throws no light upon the nature of
the process itself.

THEORIES CONCERNING PHOTOSYNTHESIS

The many theories which have been advanced concerning the nature of the
chemical changes which are involved in photosynthesis have served as the
basis for much experimental study of the problem. The following brief
summary will serve to point out the general trend of these investigations and
the present state of knowledge concerning the chemistry of photosynthesis.

Von Baeyer, in 1870, advanced the hypothesis that the first step in the
process is the breaking down of carbon dioxide into carbon monoxide and
oxygen and of water into hydrogen and oxygen; that the carbon monoxide and
hydrogen then unite to produce formaldehyde, which is immediately
polymerized to form a hexose. These theoretical changes may be represented
by the following equations:

CO2 = CO + O
1.
H2O = H2 + O
2. H2 + CO = CH2O

3. 6(CH2O) = C6H12O6

In the investigations and discussions of this hypothesis, it has been
ascertained: first, that carbon monoxide has never been found in the free form
in plant tissues; second, that when Tropaeolum plants were surrounded with an
atmosphere in which there was no carbon dioxide, but which contained
sufficient carbon monoxide to give a concentration of this gas in the cell-sap
equivalent to that in which CO2 is normally present, the plants grew normally
and apparently elaborated starch; third, other and more extensive experiments
indicated, however, that green plants in general cannot make use of carbon
monoxide gas for photosynthesis, although this does not prove that von
Baeyer's idea that CO is a step in the process is necessarily erroneous; and
finally it was shown that carbon monoxide, in sufficient concentration to
produce the results with Tropaeolum mentioned above, usually acts as a
powerful anæsthetic towards most other plants. While these considerations do
not positively prove that von Baeyer's hypothesis is incorrect, they render it so
improbable that it has generally been abandoned in favor of others which are
described below.

Erlenmeyer, even before the experimental work mentioned in the preceding
paragraph had been reported, suggested that instead of assuming a separate
breaking down of the carbon dioxide and water, it is easier to conceive that
they are united in the cell-sap into carbonic acid and that this is reduced by the
chlorophyll-containing protoplasm into formic acid and then to formaldehyde,
as indicated by the following equations:

1. H2CO3 = H2CO2 + O
2. H2CO2 = CH2O + O

Like von Baeyer's hypothesis, this assumes that formaldehyde and oxygen
are the first products of photosynthesis.

Proceeding upon this assumption, many investigators have studied the
question as to whether formaldehyde actually is present in green leaves.
Several workers have reported successful identification of formaldehyde in the
distillate from green leaves; while others have criticized these results and have
maintained that formaldehyde can likewise be obtained by distilling
decoctions of dry hay, etc., in which the photosynthetic process could not
possibly be conceived to be at work. Other investigators, notably Bach and
Palacci, reported that they had succeeded in artificially producing
formaldehyde from water and carbon dioxide, in the presence of a suitable
catalyzer or sensitizer. Euler, however, later showed conclusively that under
the conditions described by these investigators, formaldehyde can be obtained
even if no carbon dioxide is present, being apparently produced by the action
of water upon the organic sensitizer which was used.

These conflicting reports led Usher and Priestley, in a series of studies
reported between 1906 and 1911, to submit the whole matter to a critical
review. Briefly, these investigators showed that the photolysis of carbon
dioxide and water results in the formation of formaldehyde and hydrogen
peroxide, as represented by the equation

CO2 + 3H2O = CH2O + 2H2O2.

The formaldehyde is then condensed by the protoplasm into sugars, while
the hydrogen peroxide is decomposed, by an enzyme in the plant cell, into
water and oxygen. If the formaldehyde is not used up rapidly enough by the
protoplasm, it kills the enzyme and the undecomposed hydrogen peroxide
destroys the chlorophyll, which stops the whole photosynthetic process. Usher
and Priestley were able to cause the photolysis of carbon dioxide and water
into formaldehyde outside of a green plant, in the presence of a suitable
catalyzing agent which continually destroys the hydrogen peroxide as fast as it
is formed; to show the actual bleaching effect of an excess of hydrogen
peroxide in plant tissues which had been treated in such a way as to prevent

the enzyme from decomposing it; and, finally, to demonstrate the condensation
of formaldehyde into starch by the action of protoplasm which contained no
chlorophyll.

In the meantime, Fenton, in 1907, found that in the presence of magnesium
as a catalyst (it will be shown in Chapter VIII that magnesium is a constituent
of the chlorophyll molecule) formaldehyde may be obtained from a solution of
carbon dioxide in water, especially if weak bases are present.

Further, Usher and Priestley's later results showed that radium emanations,
acting upon a solution of carbon dioxide in water, produce hydrogen peroxide
and formaldehyde, and the latter polymerizes but not up to the point
represented by the hexose sugars; also, that the ultra-violet rays from a
mercury vapor lamp are very effective in bringing about the production of
hydrogen peroxide and formaldehyde from a saturated aqueous solution of
carbon dioxide, the reaction taking place even in the absence of any
"sensitizer," but much more readily if some "optical" or "chemical" sensitizer
is present. Finally, these investigators were able to duplicate all their results,
using green plant tissues, and to show that the temperature changes which take
place in a film of chlorophyll when it is exposed to an atmosphere of moist
carbon dioxide in the sunlight are such as would be required by the formation
of formaldehyde and hydrogen peroxide from carbonic acid.

More recently, Ewart has showed that formaldehyde can combine
chemically with chlorophyll; from which fact, Schryver deduces the theory
that if for any reason the condensation of formaldehyde into carbohydrates by
the cell protoplasm does not proceed as rapidly as the formaldehyde is
produced by photosynthesis, the excess of the latter enters into combination
with the chlorophyll, and that if condensation into sugar uses up all the free
formaldehyde which is present in the active protoplasm, the compound of
formaldehyde with chlorophyll is broken down setting free an additional
supply for further sugar manufacture. According to this conception there are,

in the chlorophyll-bearing protoplasm, not only the agencies for the production
of formaldehyde from carbon dioxide and water and for the condensation of
this into carbohydrates, but also a chemical mechanism by means of which the
amount of free formaldehyde in the reacting mass may be regulated so that at
no time will it reach the concentration which would be injurious to the cell
protoplasm or fall below the proper proportions for sugar-formation. This
explanation affords a satisfactory solution of the difficulty which formerly
confronted the students of photosynthesis, namely, the fact that free
formaldehyde is powerfully toxic to cell protoplasm. Without some such
conception, it was difficult to imagine how the presence of formaldehyde in
the cell contents, even as a transitory intermediate product, could be otherwise
than injurious.

As a result of these studies, the nature of the chemical changes which result
in the production of formaldehyde as the first product of photosynthesis, with
the liberation of a volume of oxygen equal to that of the carbon dioxide
consumed, seems to be fairly well established.

THE PRODUCTION OF SUGARS AND STARCHES

The next step in the process, the conversion of formaldehyde into sugars
and starches, is not necessarily a photosynthetic one, as it can be brought about
by protoplasm which contains no chlorophyll or other energy-absorbing
pigment. It is, however, a characteristic synthetic activity of living protoplasm.
There is little definite knowledge as to how the cell protoplasm accomplishes
this important task. As has been pointed out, the polymerization of
formaldehyde into a sugar-like hexose, known as "acrose," can be easily
accomplished by ordinary laboratory reactions, and acrose can be converted
into glucose or fructose by a long and difficult series of transformations. But
such processes as are employed in the laboratory to accomplish these artificial
synthesis of optically-active sugars from formaldehyde can have no relation

whatever to the methods of condensation which are used by cell protoplasm in
its easy, almost instantaneous, and nearly continuous accomplishment of this
transformation. Furthermore, these simple hexoses are by no means the final
products of cell synthesis, even of carbohydrates alone. In many plants, starch
appears as the final, if not the first, product of formaldehyde condensation. At
least, the transformation of the simple sugars, which may be supposed to be
the first products, into starch is effected so nearly instantaneously that it is
impossible to detect measurable quantities of these sugars in the
photosynthetically active cells of such plants. Other species of plants always
show considerable quantities of simple sugars in the vegetative tissues, and
some even store up their reserve carbohydrate food material in the form of
glucose or sucrose. Attempts have been made to associate the type of
carbohydrate formed in cell synthesis with the botanical families to which the
plants belong, but with no very great success. For each individual species,
however, the form of carbohydrate produced is always the same, at least under
normal conditions of growth. For example, the sugar beet always stores up
sucrose in its roots, although under abnormal conditions considerable
quantities of raffinose are developed. Similarly, potatoes always store up
starch, but with abnormally low temperatures considerable quantities of this
may be converted into sugar, which becomes starch again with the return to
normal conditions.

While it is impossible, with our present knowledge, to even guess at the
mechanism by which protoplasm condenses formaldehyde into sugars and
these, in turn, into more complex carbohydrates, the structure and relationships
to each other of the final products of photosynthesis are well known, and are
discussed at length in the following chapter.

References

Barnes, C. R.—"Physiology" (Part II of Coulter, Barnes and
Cowles' "Textbook of Botany"), 187 pages, 18 figs., Chicago,
1910.

Ganong, W. F.—"Plant Physiology," 265 pages, 65 figs., New
York, 1908 (2d ed.).

Jost, L., trans. by Gibson, R. J. H.—"Plant Physiology," 564
pages, 172 figs., Oxford, 1907.

Marchlewski, L.—"Die Chemie des Chlorophylls," 187 pages, 5
figs., 7 plates, Berlin, 1909.

Parkin, John.—"The Carbohydrates of the Foliage Leaf of the
Snowdrop (Galanthus nivalis L.) and their Bearing on the First
Sugar of Photosynthesis," in Biochemical Journal, Vol. 6, pages 1
to 47, 1912.

Pfeffer, W., trans. by Ewart, A. J.—"Physiology of Plants." Vol. I,
632 pages, 70 figs., Oxford, 1900.

CHAPTER IV

CARBOHYDRATES

These substances comprise an exceedingly important group of compounds,
the members of which constitute the major proportion of the dry matter of
plants. The name "carbohydrate" indicates the fact that these compounds
contain only carbon, hydrogen, and oxygen, the last two elements usually
being present in the same proportions as in water. As a rule, natural
carbohydrates contain six, or some multiple of six, carbon atoms and the same
number of oxygen atoms less one for each additional group of six carbons
above the first one; e.g., C6H12O6, C12H22O11, C18H32O16, etc.

Carbohydrates are classed as open-chain compounds, that is, they may be
regarded as derivatives of the aliphatic hydrocarbons. From the standpoint of
the characteristic groups which they contain, they are aldehyde-alcohols. In
common with many other polyatomic open-chain alcohols, they generally
possess a characteristic sweet, or mildly sweetish, taste. In the case of the more
complex and less soluble forms, this sweetish taste is scarcely noticeable and
these compounds are commonly called the "starches," as contrasted with the
more soluble and sweeter forms, known as "sugars."

The characteristic ending ose is added to the names of the members of this
group. As systematic names, the Latin numeral indicating the number of

carbon atoms in the molecule is combined with this ending; e.g., C5H10O5,
pentose, C6H12O6, hexose, etc.

In recent years, as a matter of scientific interest, many sugarlike substances
which contain from two to nine carbon atoms combined with the proper
number of hydrogen and oxygen atoms to be equivalent to the same number of
molecules of water in each case, have been artificially prepared in the
laboratory and designated as dioses, trioses, tetroses, pentoses, hexoses,
heptoses, octoses, and nonoses, respectively. Substances corresponding in
composition and properties with the artificial tetroses and one or two
derivatives of heptoses are occasionally found in plant tissues, and a
considerable number of pentoses and their condensation products are common
constituents of plant gums, etc.; but the great majority of the natural
carbohydrates are hexoses and their derivatives.

GROUPS OF CARBOHYDRATES

Since the simpler carbohydrates are sugars, i.e., they possess the
characteristic sweet taste, the name "saccharide" is used as a basis for the
classification of the entire group. The simplest natural sugars, the hexoses,
C6H12O6, are known as mono-saccharides. The group of next greater
complexity, those which have the formula C12H22O11 and may be regarded as
derived from the combination of two molecules of a hexose with the dropping
out of one molecule of water at the point of union, are known as di-
saccharides. Compounds having the formula C18H32O16 (i.e., three molecules
of C6H12O6 minus two molecules of H2O) are tri-saccharides; and the still
more complex groups, having the general formula (C6H10O5)n, are called the
poly-saccharides. The mono-, di-, and tri-saccharides are generally easily
soluble in water, have a more or less pronouncedly sweet taste, and are known
as the sugars; while the polysaccharides are generally insoluble in water and

of a neutral taste, and are called starches. As will be seen later, there are many
natural plant carbohydrates belonging to each of these groups.

In addition to these saccharide groups, there are other types, or groups, of
compounds which resemble the true carbohydrates in their chemical
composition and properties and are often considered as a part of this general
group. These are the pentoses, C5H10O5, and their condensation products, the
pentosans (C5H8O4)n, and their methyl derivatives, C6H12O5; certain
polyhydric alcohols having the formula C6H8(OH)6; pectose and its
derivatives, pectin and pectic acid; and lignose substances of complex
composition. It is doubtful whether these compounds are actual products of
photosynthesis in plants, or have the same physiological uses as the
carbohydrates and it has seemed wise to consider them in a separate and later
chapter.

ISOMERIC FORMS OF MONOSACCHARIDES

Four sugars having the formula C6H12O6, namely, glucose, fructose,
mannose, and galactose, occur very commonly and widely distributed in
plants. In addition to these, thirteen others having the same percentage
composition have been artificially prepared, while seven additional forms are
theoretically possible. In other words, twenty-four different compounds, all
having the same empirical formula and similar sugar-like properties are
theoretically possible. In order to arrive at a conception of this multiplicity of
isomeric forms, it is necessary to understand the two types of isomerism which
are involved. One of these is structural isomerism, and the other is space- or
stereo-isomerism.

Structural Isomerism.—This refers to an actual difference in the
characteristic groups which are present in the molecule. As has been said, all
carbohydrates, from the standpoint of the characteristic groups which they

contain, are aldehyde-alcohols. The hexoses all contain five alcoholic groups
and one primary aldehyde, or one secondary aldehyde (ketone), group. If the
aldehyde oxygen is attached to the carbon atom which is at the end of the six-

membered chain, the structural arrangement is that of an aldehyde, and
the sugar is of the type known as "aldoses"; whereas, if the oxygen is attached

to any other carbon in the chain, the ketone arrangement, results and the
sugar is a "ketose." This difference is illustrated in the Fischer open-chain
formulas for glucose (an aldose) and fructose (a ketose) as follows:

Stereo-isomerism, or space isomerism, as its name indicates, depends upon
the different arrangement of the atoms or groups in the molecule in space, and
not upon any difference in the character of the constituent groups. This
possibility depends upon the existence in the molecule of the substance in
question of one or more asymmetric carbon atoms and manifests itself in
differences in the optical activity of the compound.[1] Thus, in the formula for
glucose shown above there appear four asymmetric carbon atoms, namely,

those of the four secondary alcohol groups (in the terminal, or primary alcohol,
group, carbon is united to hydrogen by two bonds, and in the aldehyde group it
is united to oxygen by two bonds). Similarly, fructose contains three
asymmetric carbon atoms.

As an example of how the presence of these asymmetric carbon atoms
results in the possibility of many different space relationships, the following
graphic illustrations of the supposed differences between dextro-glucose and
levo-glucose, and between dextro- and levo-galactose, may be cited.[2]

Comparisons of the above formulas will show that the difference between
the formulas for d- and l-glucose lies in the arrangement of the H atoms and
the OH groups around the two asymmetric carbon atoms next the aldehyde end
of the chain; while the d- and l-galactoses differ in that this arrangement is in
the reverse order around all four of the asymmetric carbons. By similar
variations in the grouping around the four asymmetric atoms, it is possible to
produce the sixteen different space arrangements shown on page 37 for the
groups of an aldohexose. Sugars corresponding to fourteen of these different
forms have been discovered, three of which are of common occurrence in
plants, either as single mono-saccharides or as constituent groups in the more

complex carbohydrates; the remaining two forms have only theoretical
interest.

Similarly, for a ketohexose, which contains three asymmetric carbon atoms,
there are eight possible arrangements. Three sugars of this type are known,
only one (fructose) being common in plants; the others are of only theoretical
interest.

FOOTNOTES:

[1 It is assumed that the reader, or student, is familiar with the theoretical and experimental
] evidence in support of the existence of the so-called "asymmetric" carbon atom and its relation
to the effect of the compound which contains it, when in solution, in rotating the plane of polarized
light. For purposes of review, or of study of this most interesting and important phenomenon, the
reader is referred to any standard text-book on Organic Chemistry.

[2 Attention should be called, at this point, to the fact that such formulas as these cannot
] possibly accurately represent the actual arrangement of the constituent groups of a
carbohydrate molecule around an asymmetric carbon atom. The limitations of a plane-surface
formula prevent any illustration of the three-dimension relationships in space. Furthermore, there
are certain facts in connection with the birotation phenomenon and the relation of the molecular
configuration to biochemical properties (which see) that cannot be explained on the basis of the
open-chain arrangement represented by the Fischer formulas used here. A closed-ring arrangement,
showing the aldehyde oxygen as linked by its two bonds to the first and the fourth carbon atoms of
the chain, thus forming a closed-ring of four carbon and one oxygen atoms, instead of being
attached by both bonds to a single carbon atom, as in the above formulas, is undoubtedly a more
nearly accurate representation of the actual linkage in the molecule than are the open-chain
formulas used above.

The differences in conception embodied by these two types of formulas may be shown by the
following formulas for glucose:

It will be observed that in the closed-ring formula there are five asymmetric carbon atoms, and
the asymmetry of the terminal one forms the basis for the explanation of the existence of the so-
called α and β modification of d-glucose (see page 46). However, the ordinary aldehyde reactions of
the sugars are more clearly indicated by the open-chain formula. Some investigators are inclined to
be that sugars actually exist in the open-chain arrangement when in aqueous solution, and in the
closed-ring arrangement when in alcoholic solution. The closed-ring formulas will be used in this
text in the discussions of the birotation phenomena and of biochemical properties, but for the
explanations of the stereo-isomeric forms and similar phenomena, the open-chain formulas are just
as useful in conveying an idea of the possibilities of different space relationships, and are so much
simpler in appearance and in mechanical preparation, that it seems desirable to use these rather than
the more accurate closed-ring formulas.

CHEMICAL CONSTITUTION OF
MONOSACCHARIDES

The term "monosaccharides," as commonly used, refers to hexoses. It
applies equally well, however, to any other sugar-like substance which either
occurs naturally or results from the decomposition of more complex
carbohydrates, and which cannot be further broken down without destroying
its characteristic aldehyde-alcohol groups and sugar-like properties.

All such monosaccharides, being alcohol-aldehydes, can easily be reduced
to the corresponding polyatomic alcohols, containing the same number of
carbon atoms as the original monosaccharides, each with one OH group

attached to it. All aldose monosaccharides are converted, by gentle oxidation,
into the corresponding monobasic acid, having a COOH group in the place of
the original CHO group. Further oxidation either changes the alcoholic groups
into COOH groups, producing polybasic acids, or breaks up the chain. When
ketose monosaccharides are submitted to similar oxidation processes, they are
broken down into shorter chain compounds.

The various monosaccharides which have thus far been found as
constituents of plant tissues, or as parts of other more complex compounds
which occur in plants, are shown in the following table:

Trioses (C3H6O3) Tetroses (C4H8O4)
Aldose— Glyceric aldehyde, or Aldoses d- and l-
glycerose — Erythrose,
l-Threose
Ketose— Dioxyacetone

Pentoses (C5H10O5) Methyl Pentoses (C6H12O5)
Aldoses d- and l-Arabinose Aldoses Rhamnose
— —
d- and l-Xylose Fucose
l-Ribose Rhodeose
l-Lyxose Chinovose

Hexoses (C6H12O6)
Mannitol series Dulcitol series
Aldoses d- and l-Glucose d- and l-
— Galactose
d- and l-Mannose d- and l-Talose
d- and l-Gulose
d- and l-Idose
d-Altrose
d-Allose
Ketoses
d-Fructose d-Tagatose
—

d-Sorbose

Heptoses Octoses Nonoses
(C7H14O7) (C8H16O8) (C9H18O9)
Glucoheptose Gluco-octose Glucononose
Mannoheptose Manno-octose Mannononose
Galactoheptose Galacto-octose
Persuelose
Sedoheptose

The hexoses are by far the most important group of monosaccharides. They
are undoubtedly the first products of photosynthesis, and all the other
carbohydrates may be considered to be derived from them by condensation.
Because of their biochemical significance and their immense importance as the
fundamental substances for all plant and animal energy-producing materials,
the following detailed studies of their chemical composition and molecular
configuration are fully warranted.

That all the hexoses contain five alcoholic groups is proved by the
experimental evidence that each one forms a penta-ester, by uniting with five
acid radicals, when treated with mineral or organic acids under proper
conditions. Thus, glucose penta-acetate, penta-nitrate, penta-benzoate, etc.,
have all been prepared. The presence of the aldehyde group is proved by the
fact that all aldohexoses have been converted, by gentle oxidation, into
pentaoxy-monobasic acids, and the ketohexoses broken down into shorter
chain compounds by similar gentle oxidations; these reactions being
characteristic of compounds containing an aldehyde and a ketone group
respectively. This experimental evidence establishes the nature of the
characteristic groups in the molecule, in each case.

The molecular configurations illustrated in the following table are those
suggested by Emil Fischer, as a result of his exhaustive studies of the chemical
constitution of the various carbohydrates. There is, of course, no thought that

the printed formulas here presented accurately represent the actual
relationships in space of the different groups; but there is fairly conclusive
evidence that the variations in special groupings in the different sugars are
properly referable to the particular asymmetric carbon atoms as indicated in
the several formulas as presented.

Reference will be made in subsequent paragraphs to the probable chemical
constitution of the monosaccharides other than hexoses; but the above
discussion of the structure of the hexoses will serve as a sufficient introduction
to the study of the composition of the common carbohydrates.

CHARACTERISTIC REACTIONS OF HEXOSES

Specific Rotatory Power.—All soluble carbohydrates, since they contain
asymmetric carbon atoms, with the consequent larger groups on one side of the
molecule than the other, rotate the plane of polarized light when it passes
through a solution of the carbohydrate in question. The amount of the rotation
depends upon the nature of the carbohydrate, the concentration of the solution,

and the length of the column of solution through which the ray of polarized
light passes. But the same definite amount of the same sugar, dissolved in the
same volume of water, and placed in a tube of the same length, will always
cause the same angular deviation, or rotation, of the plane in which the
polarized light which passes through it is vibrated. In other words, the same
number of molecules of the optically active substance in solution will always
produce the same rotatory effect. This is called the specific rotatory power of
the substance in question. It is expressed as the number of degrees of angular
deviation of the plane of polarized light caused by a column of the solution
exactly 200 mm. in length, the concentration of the solution being 100 grams
of substance in 100 cc. at a temperature of 20° C. Actual determinations of
specific rotatory power are usually made with solutions more dilute than this
standard, and the observed deviation multiplied by the proper factor to
determine the effect which would be produced by the solution of standard
concentration. If the direction of the deviation is to the right (i.e., in the
direction in which the hands of the clock move) it is spoken of as "dextro"
rotation and is indicated by the sign +, or the letter d; while if in the opposite
direction, it is called "levo" rotation and indicated by the sign -, or the letter l.
For example, the specific rotation of ordinary glucose is +52.7°; of fructose,
-92°; of sucrose, +66.5°.

Reducing Action.—All of the hexose sugars are active reducing agents.
This is because of the aldehyde group which they contain. Many of the
common heavy metals, when in alkaline solutions, are strongly reduced when
boiled with solutions of the hexose sugars. Alkaline copper solutions yield a
precipitate of red cuprous oxide; ammoniacal silver solutions give silver
mirrors; alkaline solutions of mercury salts are reduced to metallic mercury,
etc. Any sugar which contains a potentially active aldehyde group will exhibit
this reducing effect and is known as a "reducing sugar." In some of the di- and
tri-saccharides, the linkage of the hexose components together is through the
aldehyde group, in such a way that it loses its reducing effect; such sugars are
known as "non-reducing." Advantage is taken of this property for both the

detection and quantitative determination of the "reducing sugars." A standard
alkaline copper solution of definite strength, known as "Fehling's solution," is
added to the solution of the sugar to be tested and the mixture boiled, when the
characteristic brick-red precipitate appears. If certain standard conditions of
volume of solutions used, length of time of boiling, etc., are observed, the
quantity of cuprous oxide precipitated bears a definite ratio to the amount of
sugar which is present, so that if the precipitate be filtered off and weighed
under proper conditions, the weight of sugar present in the original solution
can be calculated. The proper conditions for carrying on such a determination
and tables showing the amounts of the various "reducing sugars" which
correspond to the weight of cuprous oxide found, are given in all standard text-
books dealing with the analysis of organic compounds.

Fermentability.—The common hexoses are all easily fermented by yeast,
forming alcohol and carbon dioxide, according to the equation

C6H12O6 = 2C2H5OH + 2CO2.

The importance and biochemical significance of this reaction will be
considered in detail in connection with the discussions of the relation of
molecular configuration to biochemical properties (see page 56) and the nature
of enzyme action (see page 194).

Formation of Hydrazones and Osazones.—Another property of the
hexoses which is due to the presence of an aldehyde group in the molecule, is
that of forming addition products with phenyl hydrazine, known as
"hydrazones" and "osazones." For example, glucose reacts with phenyl
hydrazine in acetic acid solution, in two stages. The first, which takes place
even in a cold solution may be represented by the equation

C6H12O6 + C6H5·NH·NH2 = C6H12O5:N·NH·C6H5 + H2O.
Glucose Phenyl-hydrazine Glucose-hydrazone

The structural relationships involved may be represented as follows:

The hydrazones of the common sugars, with the exception of the one from
mannose, are colorless compounds, easily soluble in water. Hence, they do not
serve for the separation or identification of the individual sugars. But if the
solution in which they are formed contains an excess of phenyl hydrazine and
is heated to the temperature of boiling water for some time, the alcoholic
group next to the aldehyde group (the terminal alcohol group in ketoses) is
first oxidized to an aldehyde and then a second molecule of phenyl hydrazine
is added on, as illustrated above, forming a di-addition-product, known as an
"osazone." The osazones are generally more or less soluble in hot water, but on
cooling they crystallize out in yellow crystalline masses each with definite
melting point and crystalline form. All sugars which have active aldehyde
groups in the molecule form osazones. These afford excellent means of
identification of unknown sugars, or of distinguishing between sugars of
different origin and type.

Glucose, mannose, and fructose all form identical osazones. This is because
the structure of these three sugars is identical except for the arrangement
within the two groups at the aldehyde end of the molecule (see formulas on
page 44). Since it is to these two groups that the phenyl hydrazine residue
attaches itself, it follows that the resulting osazones must be identical in
structure and properties. All other reducing sugars yield osazones of different
physical properties.

When an osazone is decomposed by boiling with strong acids, the phenyl
hydrazine groups break off, leaving a compound containing both an aldehyde
and a ketone group. Such compounds are known as "osones." The osones from
glucose, mannose, and fructose are identical. By carefully controlled
reduction, either one of the C=O groups of the osone may be changed to an
alcoholic group, producing thereby one of the original sugars again. Hence, it
is possible to start with one of these sugars, convert it into the osone and then
reduce this to another sugar, thereby accomplishing the transformation of one
sugar into another isomeric sugar.

Formation of Glucosides.—By treatment with a considerable variety of
different types of compounds, under proper conditions, it is possible to replace
one of the hydrogen atoms of the terminal alcoholic group of the hexose
sugars with the characteristic group of the other substance, forming
compounds known, respectively, as glucosides, fructosides, galactosides, etc.
The structural relation of methyl glucoside to glucose, for example, may be
illustrated as follows:

A general formula for glucosides is R·(CHOH)5·CHO; and the R may
represent a great variety of different organic radicals (see the chapters dealing
with Glucosides and with Tannins). When the glucosides are hydrolyzed, they
yield glucose and the hydroxyl compound of the radical with which it is

united. All the statements which have been made with reference to glucosides,
apply equally well with reference to fructosides, galactosides, mannosides, etc.

It is possible, by various laboratory processes, to replace additional
hydrogen atoms in the glucose molecule with the same or other organic
radicals, thus producing glucosides containing two or more R groups; but most
of the natural glucosides contain only one other characteristic group.

Oxidations.—When the hexoses are oxidized they give rise to three
different types of acids, depending upon the conditions of the oxidation and
the kind of oxidizing agent used. With glucose, for example, the relationships
involved may be illustrated as follows:

An important property of the acids of the gluconic type is that when heated
with pyridine or quinoline to 130°-150° they undergo a molecular
rearrangement whereby the acid corresponding to an isomeric sugar is
produced. For example, gluconic acid, under these conditions, becomes
mannonic acid, which can be reduced to mannose. The process is reversible;
mannose can be converted to mannonic acid, thence to gluconic acid, thence to
glucose. Similarly, galactonic acid can be converted into talonic acid, and this
to talose, and this process is reversible. These facts afford another means of
conversion of one sugar into another.

From the standpoint of physiological processes, glucuronic acid is the most
interesting and important oxidation product of glucose. It is often found in the
urine of animals, as the result of the partial oxidation of glucose in the animal
tissues. Normally, glucose is oxidized in the body to its final oxidation

products, carbon dioxide and water. But when many difficultly oxidizable
substances, such as chloral, camphor, turpentine oil, aniline, etc., are
introduced into the body, the organism has the power of combining these with
glucose to form glucosides. These so-called "paired" compounds are then
oxidized to the corresponding glucuronic acid derivatives and eliminated from
the body in the urine. No phenomenon similar to this occurs in plants,
however, and glucuronic acid has never been found in plant tissues.

Synthesis and Degradation of Hexoses.—Monosaccharides of any desired
number of carbon atoms can be produced from aldoses having one less carbon
atoms, by way of the familiar "nitrile" reaction. Aldoses, like all other
aldehydes, combine directly with hydrocyanic acid, forming compounds
known as nitriles, which contain one more carbon atom than was present in the
original aldehyde; the cyanogen group can easily be converted into a COOH
group; and this, in turn, reduced to an aldehyde, thus producing an aldose with
one more carbon atom than was present in the initial sugar. These changes may
be illustrated by the following equations:

It is possible, by this process, to advance step by step from formaldehyde to
higher sugars, Emil Fischer and his students having carried the process as far
as the production of glucodecose (C10H20O10). It usually happens, however,
that two stereo-isomers result from the "step-up" by way of the nitrile reaction;
thus, arabinose yields a mixture of glucose and mannose, glucose yields
glucoheptose and mannoheptose, etc.

The reverse process, or the so-called "degradation" of a sugar into another
containing fewer carbon atoms, may be readily accomplished in either one or
two ways. In Wohl's process, the aldehyde group of the sugar is first converted
into an oxime, by treatment with hydroxylamine; the oxime, on being heated
with concentrated sodium hydroxide solution, splits off water and becomes the
corresponding nitrile; this, on further heating, splits off HCN and yields an
aldose having one less carbon atom than the original sugar. This process is the
exact reverse of the nitrile synthesis, described above. The second method of
degradation, suggested by Ruff, makes use of Fenton's method of oxidizing

aldehyde sugars to the corresponding monobasic acid, using hydrogen
peroxide and ferrous sulfate as the oxidizing mixture; the aldonic acid thus
formed is then converted into its calcium salt, which, when further oxidized,
splits off its carboxyl group and one of the hydrogens of the adjacent alcoholic
group, leaving an aldose having one less carbon atom than the original aldose
sugar.

Enolic Forms.—A final avenue for the interconversion of glucose,
mannose, and fructose into one another, is through the spontaneous
transformations which these undergo when dissolved in water containing
sodium hydroxide or potassium hydroxide. This change is due to the
conversion of the sugar, in the alkaline solution, into an enol, which is
identical for all three sugars, and which may subsequently be reconverted into
any one of the three isomeric hexoses. The relationships involved are
illustrated in the following formulas:

The preceding technical discussion of the chemical constitution and
reactions of the hexoses has been presented, not because it has any direct
connection with the occurrence or functions of these compounds in plant
tissues, but for the purpose of giving to the student a graphic conception of the
structure and properties of these simple carbohydrates, as a basis for the

understanding of the nature, properties, possible chemical reactions, syntheses,
etc., of the more complex types of carbohydrates, which, along with these
simple monosaccharides, constitute the most important single group of organic
components of plants.

THE OCCURRENCE AND PROPERTIES OF
MONOSACCHARIDES

Only two monosaccharides occur as such in plants. These are glucose and
fructose. All the other hexoses, whose structure is shown on pages 37 and 38,
occur in plants only as constituents of the more complex saccharides, in
glucoside-formations, or as the corresponding polyatomic alcohols.

The aldo-hexoses which occur most commonly in plants, either free or in
combination, are d-glucose, d-mannose, and d-galactose; while d-fructose and
d-sorbose are the common keto-hexoses.

Glucose (often called also dextrose, fruit sugar, or grape sugar) occurs
widely distributed in plants, most commonly in the juices of ripening fruits,
where it is usually associated with fructose and sucrose, the two hexoses being
easily derived from sucrose by hydrolysis. Glucose is also produced by the
hydrolysis of many of the more complex carbohydrates, by the action either of
enzymes or of dilute acids; lactose, maltose, raffinose, starch, and cellulose, as
well as many glucosides all yielding glucose as one of the products of their
hydrolysis. In all such cases, it is d-glucose which is obtained.

Glucose is a crystalline solid (although it does not form such sharply
defined crystals as does sucrose, or "granulated sugar"), which is easily soluble
in water. It usually appears on the market in the form of thick syrups, which
are produced commercially by the hydrolysis of starch with dilute sulfuric
acid, removal of the acid after the hydrolysis is complete, and evaporation of

the resulting solution to the desired syrupy consistency. (Since corn starch is
commonly used as the raw material for this process, these syrups are often
spoken of as "corn syrup.") The sweetness of glucose is about three-fifths that
of ordinary cane sugar.

Glucose exhibits all the properties of hexoses which have been described in
general terms above. It is a reducing-sugar, and is easily fermented. The
specific rotatory power of d-glucose is +52.7°. But when glucose is dissolved
in water, it exhibits in a marked degree the phenomenon known as
"mutarotation"; that is, freshly made solutions exhibit a certain definite
rotatory power, but this changes rapidly until it finally reaches another definite
specific rotation. In other words, glucose is "birotatory," or possesses two
distinct specific rotatory powers, and the changing rotation effect in aqueous
solutions is due to the change from one form to the other. When dissolved in
alcohol, it does not exhibit this change in rotatory power. In order to explain
this phenomenon, it is necessary to assume that there are two modifications of
d-glucose, which have been designated respectively as the α and β forms. The
possibility of the existence of these two forms is explained by the assumption
of the closed-ring arrangement of the glucose molecule, as indicated in the
following formulas which represent the two possible isomeric arrangements:

It is assumed that the α modification (with its specific rotatory power of
+105°) is the normal form for crystalline glucose, but that when dissolved in
water it is changed into an aldehydrol, i.e., a compound containing two
additional OH groups, which later breaks down again, into the β modification
(with its specific rotatory power of +22°). When dissolved in alcohol, this
change does not take place because of the absence of the excess of water
necessary to produce the intermediate aldehydrol form.

There are other examples of the existence of the α and β modification of
glucose. For example, α-methyl-glucoside and β-methyl-glucoside (specific
rotatory powers, +157° and -33°, respectively) are both known, as well as
several other similar glucoside arrangements.

Mannose.—This sugar does not occur as such in plants; but complex
compounds which yield d-mannose when hydrolyzed, known as "mannosans,"
are found in a number of tropical plant forms. The mannose which is obtained

from these by hydrolysis is very similar to glucose in its properties, forms the
same osazones as do glucose and fructose, exhibits mutarotation, etc. Mannose
may also be obtained by oxidizing mannitol, a hexatomic alcohol, known as
"mannite," which occurs in many plants, especially in the manna-ash
(Fraxinus ornus), the dried sap from which is known as "manna."

Galactose occurs in the animal kingdom as one of the constituents of
lactose, or milk-sugar. It is also one of the constituents of raffinose, a
trisaccharide sugar found in plants, and occurs as "galactans" in many gums
and sea-weeds. The d-galactose, obtained by the hydrolysis of any of these
compounds, is a faintly sweet substance which resembles glucose in many of
its properties; having one characteristic difference, however, in that it forms
mucic acid instead of saccharic acid when oxidized by concentrated nitric acid.
These oxidation products are very different in their physical properties and this
difference serves to distinguish between the two sugars from which they are
derived.

Fructose (levulose, honey sugar, or "diabetic" sugar) occurs along with
glucose in the juices of many fruits, etc. It is a constituent of sucrose, of
raffinose, and of the polysaccharide inulin, from which it may be obtained by
hydrolysis. It is a ketose sugar, reduces Fehling's solution, forms the same
osazone as glucose, and is easily fermentable by yeast. Its sweetness is slightly
greater than that of ordinary cane sugar. d-fructose (the ordinary form) is easily
soluble in water, and is strongly levorotatory, its specific rotatory power at 20°
C. being -92.5°; it is unique in the very large effect which is produced in its
rotatory power by increasing the temperature of the solution; at 87° its specific
rotatory power is reduced to -52.7°, exactly equal to but in the opposite
direction of the effect of glucose; hence, invert sugar, which is a mixture of an
equal number of molecules of glucose and fructose, and which has a specific
rotatory power of -19.4° at 20° C., becomes optically inactive at 82° C.

Sorbose is the only other ketohexose which has any importance in plant
chemistry. It does not occur free in plants, but is the first oxidation product
from the hexatomic alcohol, sorbitol, which is present in the juice of the
berries of the mountain-ash. Sorbose is a crystalline solid, which is not
fermentable by yeast, but which otherwise closely resembles fructose.

DISACCHARIDES

The disaccharides, having the formula C12H22O11, may be regarded as
derived from the monosaccharides by the linking together of two hexose
groups with the dropping out of a molecule of water, in the same way that
many other organic compounds form such linkages. That this is a perfectly
correct conception, is shown by the fact that, when hydrolyzed, the
disaccharides break down into two hexose sugars, thus

C12H22O11 + H2O = C6H12O6 + C6H12O6.

With all known disaccharides, at least one of the hexoses obtained by
hydrolysis is glucose; hence all disaccharides may be regarded as glucosides
(C6H12O5·R) in which the R is another hexose group.

Since hexoses have both alcoholic and aldehyde groups, and since either of
these types of groups may function in the linkage of the two hexoses to form a
disaccharide, it is possible for two hexoses, both of which are reducing sugars
to be linked together in three different ways: (1) through an alcoholic group of
each hexose, (2) through an alcoholic group of one and the aldehyde group of
the other, and (3) through the aldehyde group of each hexose. Disaccharides
linked in either of the first two ways will be reducing sugars, since they still
contain a potentially active aldehyde group; but those of the third type will not
be reducing sugars, since the linkage through the aldehyde groups destroys
their power of acting as reducing agents. Examples of each of these three types

of linkage are found among the common disaccharides, as will be pointed out
below.

The following table shows the general characteristics of the common
disaccharides.

Type 1.—Aldehyde group potentially active, reducing sugars:
Sugar Components
Maltose Glucose and glucose
Gentiobiose Glucose and glucose
Lactose Glucose and galactose
Melibiose Glucose and galactose
Turanose Glucose and fructose

Type 2.—Non-reducing sugars:
Sucrose Glucose and fructose
Trehalose Glucose and glucose

The disaccharides of Type 1 reduce Fehling's solution and form
hydrazones and osazones, although somewhat less readily than do the
hexoses. They all show mutarotation and exist in two modifications,
indicating that the component groups have the closed-ring arrangement.

The disaccharides of Type 2, since they contain no potentially active
aldehyde group, do not reduce Fehling's solution, nor form osazones; neither
do they exhibit mutarotation. The only disaccharides which occur as such in
plants are of this type. Disaccharides of Type 1 may be obtained by the
hydrolysis of other, more complex, carbohydrates.

All disaccharides are easily hydrolyzed into mixtures of their component
hexoses, by boiling with dilute mineral acids, or by treatment with certain
specific enzymes which are adapted to the particular disaccharide in each
case (see page 55, also Chapter XIV).

Sucrose (cane sugar, beet sugar, maple sugar) is the ordinary "granulated
sugar" of commerce. It occurs widely distributed in plants, where it serves as
reserve food material. It is found in largest proportions in the stalks of sugar
cane, in the roots of certain varieties of beets, and in the spring sap of maple

trees, all of which serve as industrial sources for the sugar. In the sugar cane,
and beet-roots, it constitutes from 12 to 20 per cent of the green weight of
the tissue and from 75 to 90 per cent of the soluble solids in the juice which
can be expressed from it. Its universal use as a sweetening agent is due to the
combined facts that it crystallizes readily out of concentrated solutions and,
hence, can be easily manufactured in solid form, and that it is sweeter than
any other of the common sugars except fructose.

Sucrose is a non-reducing sugar, forms no osazone, and is not directly
fermentable by yeast, although most species of yeasts contain an enzyme
which will hydrolyze sucrose into its component hexoses, which then readily
ferment.

When hydrolyzed by acids, or by the enzyme "invertase," it yields a
mixture of equal quantities of glucose and fructose. Sucrose is
dextrorotatory, but since fructose has a greater specific rotatory action to the
left than glucose has to the right, the mixture resulting from the hydrolysis of
sucrose is levorotatory. Since the hydrolysis of sucrose changes the rotatory
effect of the solution from the right to the left, the process is usually called
the "inversion" of sucrose, and the resultant mixture of equal parts of
glucose and fructose is called "invert sugar." As has been pointed out,
solutions of invert sugar become optically inactive when heated to 82 °C.,
because of the reduction in the rotatory power of fructose due to the higher
temperature.

The probable linkage of the two hexoses to form sucrose, in such a way as
to produce a non-reducing sugar, is illustrated in the following formula:

Trehalose seems to serve as the reserve food for fungi in much the same
way that sucrose does for higher plants. It is composed of two molecules of
glucose linked together through the aldehyde group of each, as trehalose is a
non-reducing sugar. This linkage is illustrated in the following formula:

Trehalose may be hydrolyzed into glucose by dilute acids and by the
enzyme "trehalase," which is contained in many yeasts and in several species
of fungi. It is strongly dextrorotatory (specific rotatory power, +199°). It is
not fermentable by yeast.

Trehalose appears to replace sucrose in those plants which contain no
chlorophyll and do not elaborate starch. The quantity of trehalose in such
plants reaches a maximum just before spore formation begins. Since it is
manufactured in the absence of chlorophyll, its formation must be
accomplished by some other means than photosynthesis, yet it is composed
wholly of glucose—a natural photosynthetic product.

Maltose rarely occurs as such in plants, although its presence in the cell-
sap of leaves has sometimes been reported. It is produced in large quantities
by the hydrolysis of starch during the germination of barley and other grains.
This hydrolysis is brought about by the enzyme "diastase," which is present
in the sprouting grain.

Maltose is easily soluble in water, and crystallizes in masses of slender
needles. It is a reducing sugar; readily forms a characteristic osazone; is
strongly dextrorotatory (specific rotatory power +137°); and is readily
fermented by ordinary brewer's yeast, which contains both "maltase" (the
enzyme which hydrolyzes maltose to glucose) and "zymase" (the alcohol-
producing enzyme). When hydrolyzed, either by dilute acids or by maltase,
one molecule of maltose yields two molecules of glucose. Its component
hexoses are, therefore, the same as those of trehalose, a non-reducing sugar,
this difference in properties being due to the difference in the point of
linkage between the two glucose molecules, that for maltose being such as to
leave one of the aldehyde groups potentially active, as shown in the
following formula,

Isomaltose is a synthetic sugar, obtained by Fischer, by condensing two
molecules of glucose. Its properties are quite similar to those of maltose, but
it yields a slightly different osazone and is not fermentable by yeast. These
differences are explained by the assumption that this sugar is a glucose-β-
glucoside, while normal maltose is a glucose-α-glucoside.

Gentiobiose is a disaccharide which results from the partial hydrolysis of
the trisaccharide gentianose (see page 53). It is very similar in its general
properties to isomaltose. Cellobiose is a disaccharide which results from the
hydrolysis of cellulose. It is a reducing sugar, forms an osazone, and
resembles maltose.

Maltose, isomaltose, gentiobiose, and cellobiose, are all glucose-
glucosides, the difference between them being undoubtedly due to linkage
being between different alcoholic groups in the glucose molecules.

The disaccharide lactose is a glucose-galactoside. It is the sugar which is
present in the milk of all mammals. It has never been found in plants.
Melibiose, which is the corresponding vegetable glucose-galactoside, may
be obtained by the partial hydrolysis of the trisaccharide raffinose (see

below). It is a reducing sugar; forms a characteristic osazone; and exhibits
mutarotation. It is not fermented by ordinary top-yeasts, but is first
hydrolyzed and then fermented by the enzymes present in bottom-yeasts.

TRISACCHARIDES

Trisaccharides, as the name indicates, consist of three hexoses (or
monosaccharides) linked together by the dropping out of two molecules of
water. Their formula is C18H32O16. When completely hydrolyzed, they yield
three molecules of monosaccharides; when partially hydrolyzed, one each of
a disaccharide and a monosaccharide.

One trisaccharide of the reducing sugar type, namely rhamnose, exists in
plants as a constituent of the glucoside xanthorhamnin. It is composed of one
molecule of glucose united to two molecules of rhamnose (methyl pentose,
C6H12O5). It is of interest only in connection with the properties of the
glucoside in which it is present (see page 84).

Three trisaccharides which are non-reducing sugars are found in plants;
namely, raffinose, gentianose, and melizitose.

Raffinose occurs normally in cotton seeds, in barley grains, and in manna;
also, in small quantities in the beet root, associated with sucrose. It is more
soluble in water than is sucrose and hence remains in solution in the
molasses from beet-sugar manufacture, which constitutes the commercial
source for this sugar. Raffinose crystallizes out of concentrated solutions,
with five molecules of water of crystallization, in clusters of glistening
prisms. It is strongly dextrorotatory, the anhydrous sugar having a specific
rotatory power of +185°, and the crystalline form, C18H32O16, showing a

specific rotation of +104.5°. It does not reduce Fehling's solution, nor form
an osazone, and in its other properties it closely resembles sucrose.

The hydrolysis of raffinose presents several interesting possibilities. If its
structure is represented as follows:

C6H11O5——C6H10O4——C6H11O5
Fructose Glucose Galactose
\_____ _____/ \_____ _____/
\/ \/
Sucrose Melibiose

it is apparent that it may break down by hydrolysis in three different ways:
(1) into sucrose and galactose, (2) into fructose and melibiose, and (3) into
fructose, glucose, and galactose. As a matter of fact, it does actually break
down in these three different ways, under the influence of different catalysts;
invertase or dilute acids break it down into fructose and melibiose, emulsin
hydrolyzes it to sucrose and galactose, while strong acids or the enzymes of
bottom-yeasts break it down into the three hexoses.

Gentianose, a trisaccharide found in the roots of yellow gentian
(Gentiana lutea), is a non-reducing sugar, which when hydrolyzed yields
either fructose and gentiobiose, or fructose and two molecules of glucose.

Melizitose, a trisaccharide which, in crystallized form, has the formula,
C18H32O16·2H2O, occurs in the sap of Larix europea and in Persian manna,
and has recently been found in considerable quantities in the manna which
collects on the twigs of Douglas fir and other conifers. When hydrolyzed, it
yields one molecule of fructose and one of turanose, a disaccharide
containing fructose and glucose linked together in a slightly different way
than they are in sucrose. Turanose itself is a reducing sugar, but when linked

with fructose to form melizitose its reducing properties are destroyed.
Melizitose is a very sweet sugar.

TETRASACCHARIDES

A complex saccharide, known as stachyose, which is found in the tubers
of Stachys tuberifera, is said by some investigators to be a tetrasaccharide
and by others to have the formula C36H62O31·7H2O (i.e., a hexasaccharide).
It is a crystalline solid, with a faintly sweetish taste, and a specific rotatory
power of +148°. When hydrolyzed it yields glucose, fructose, and two (or
more) molecules of galactose.

THE RELATION OF THE MOLECULAR
CONFIGURATION OF SUGARS TO THEIR
BIOCHEMICAL PROPERTIES

As will be pointed out later (see Chapter XIV), all chemical reactions
which are involved in vital phenomena, including those of plant growth and
metabolism, are controlled by enzymes. The biochemical reactions which
the soluble carbohydrates undergo afford such excellent illustrations of the
relation of the molecular configuration of an organic compound to the
possibility of the action of an enzyme upon it, that it seems desirable to
discuss this relationship at this point, rather than to postpone it until after the
nature of enzyme action has been considered. Undoubtedly, the student, after
he has studied the nature of enzymes and their mode of action, as presented
in Chapter XIV, will find it profitable to return to this section and review the
facts here presented, as illustrating the principles and mechanism of enzyme
action. But a consideration, at this time, of the relation of the molecular

configuration of the sugars to their biochemical reactions cannot fail to add
interest to the study of these matters from the chemical and biological
standpoints.

It has been known for a long time that the dextro- and levo-isomers of a
compound which contains one or more asymmetric carbon atoms are
affected differently by biological agents, such as yeasts, moulds, bacteria,
etc. Pasteur, as early as 1850, showed that the green mould, Penicillium
glaucum, when growing in solutions of racemic acid (a mixture of equal
molecules of d- and l-tartaric acids) uses up only the d-acid, leaving the l-
form absolutely untouched. Later, it was found that the same green mould
attacks l-mandelic acid in preference to the d- form; whereas the yeast,
Saccharomyces ellipsoideus, exhibits the opposite preference for these acids.

These observations upon some of the earlier known forms of optically
active organic acids led the way to a general study of this phenomenon as
exhibited by the optically active soluble carbohydrates. The results of these
studies may be considered in connection with the several different types of
reactions which these sugars undergo, as follows:

Glucoside Hydrolysis.—As was pointed out in connection with the
discussion of the mutarotation of glucose, this sugar may exist in either the α
or the β modification. Glucosides of both α and β glucose are of common
occurrence. The difference in molecular configuration, in such cases, may be
represented by the following formulas:

The radical represented by the R may be either a common alkyl radical (as
CH3, C2H5, etc.), another saccharide group (as in the case of the
disaccharides, trisaccharides, etc.), or some other complex organic group (as
in the case of the natural glucosides described in Chapter VI). But, in every
case, the glucoside is easily hydrolyzed by the enzyme maltase (or α-
glucase) if the molecular arrangement is that represented by the α-
attachment, or by the enzyme emulsin (or β-glucase) if the glucoside is of
the β type; but emulsin is absolutely without effect upon α-glucosides, and
maltase does not produce the slightest change in β-glucosides. These
statements hold true regardless of the nature of the group which is
represented by the R in the formulas above. Hence, the biochemical
properties of the glucosides, so far as their hydrolysis by the enzymes which
are present in many biological agents is concerned, depends wholly upon the
molecular configuration of the glucose itself. Furthermore, neither the
mannosides, which differ from glucosides only in the arrangement of the H
and OH groups attached to one of the asymmetric carbon atoms in the

hexose, nor galactosides in which two such arrangements are different (see
configuration formulas on page 57), are attacked by either maltase or
emulsin. But other enzymes specifically attack other disacharides, or
polysaccharides, or glucoside-like complexes. For example, lactase acts
energetically upon ordinary lactose and all other β-galactosides; but not upon
any glucoside, mannoside, etc.

Again, neither α- nor β-xylosides, which correspond with the above-
described glucosides in every particular except that the HCOH group next
the terminal CH2OH group is missing, are hydrolyzed by either emulsin or
maltase.

These instances, selected from among many similar observations, clearly
prove that not only the number and kind of groups in the molecule, but also
the arrangement of the constituent groups in space, must be identical in order
that the compound may be acted upon by any given enzyme acting as a
biological hydrolytic agent.

Fermentability.—The enzyme zymase, present in all yeasts, promotes the
fermentation of the natural d- forms of the three hexoses, glucose, mannose,
and fructose, but is without effect upon the artificial l- forms of the same
sugars. The uniform action of zymase upon these hexoses is easily explained
upon the basis of the same assumption which was used to account for the
formation of identical osazones from these sugars and their easy
transformation into each other; namely, their easy transformation into an
enolic form which is identical for all three.

Further, galactose is fermented by some yeasts (although not by all), but
much less readily than are the other sugars, and the temperature reaction is
quite different with galactose than with the others. Talose and tagatose are
entirely unfermentable. A study of the configuration formulas for these

several sugars shows the explanation for these observed facts. These
formulas are as follows:

It will be noted that in the case of glucose, mannose, and fructose, the
configuration is identical at every point except at the aldehyde end of the
chain, and that here the two groups readily arrange themselves into the same
enolic form for the three sugars. Galactose differs from these three sugars
only in the arrangement of the H and OH groups attached to one of the other
carbon atoms (the third from the alcoholic end); the difficulty of its
fermentation indicates that some molecular rearrangement to bring this
group into its proper configuration must precede the fermentation process.

The fact that it is the third HCOH group which thus undergoes
rearrangement is significant because of the participation of these parts of
molecules in groups of threes in many biological processes, as will be
mentioned elsewhere. Talose is unfermentable, even though the arrangement
of its upper three groups is the same as in the galactose and the lower three
the same as in mannose.

If further proof that fermentability depends upon molecular configuration
were needed, it is furnished by the fact that no pentose is fermentible, even
though the stereo-arrangement of each of the four alcoholic groups in the
molecule is identical with the corresponding groups in a fermentible hexose.

Oxidation by Bacteria.—The bacillus Bacterium xylinum contains an
enzyme, or enzymes, which promote the oxidation of the aldehyde group of
an aldose sugar to COOH, or of one alcoholic CHOH group next the
terminal CH2OH group of a hexatomic alcohol to C=O. But these oxidizing
enzymes affect only those compounds in which the OH groups are on the
same side of the two asymmetric carbon atoms next the end of the molecule
where the oxidation takes place, as indicated in the following groupings.

The configuration of the remainder of the molecule is immaterial to action
by these oxidizing bacteria; hence, the enzymes in this case are apparently
concerned only with the configuration arrangement of a portion of the
molecule, instead of with the whole hexose grouping, as in the cases of the
other reactions which have been thus far considered.

It is apparent from these illustrations, and from many more which might
be cited, that there is a very definite relation between the molecular
configuration of a carbohydrate and its biochemical properties, as
represented by the possibilities of the action of enzymes upon it. The
probable nature of this relationship will be better understood after the
general questions involved in the mode of enzyme action have been
considered (see Chapter XIV). But for the present, it will be sufficient to
note that it seems to be necessary that the enzyme shall actually fit the
molecular arrangement of the compound at all points, in the same way that a
key fits its appropriate lock; or a still better illustration is that of the fitting of
a glove to the hand. On the basis of the latter illustration, it is just as
impossible for a dextro-enzyme to affect a levo-sugar, or for α-glucase to
affect a β-glucoside, as it is to fit a right-hand glove upon a left hand. Further
attention will be given to these matters in later chapters.

POLYSACCHARIDES

The polysaccharides which, like the simpler saccharides, or sugars, which
have thus far been studied, undoubtedly serve as reserve food for plants, are
known under the general name of "starches." They are substances of high
molecular weight, whose constitution is represented by the general formula
(C6H10O5)n. It should be noted that an exactly accurate formula should be
(C6)n(H12O6)n-1; but since the value of n is very high, the simpler formula is
approximately correct. The value of n has not been accurately determined for
any of the individual members of the group, but is probably never less than
30 and may often be 200 or more. The fact that these compounds are
insoluble in most of the solvents which can be used for molecular weight
determinations makes it difficult to determine their actual molecular
constitution.

When completely hydrolyzed, the polysaccharides yield only hexoses.
They are, therefore, technically known as "hexosans." Each individual
polysaccharide which has been studied thus far yields only a single hexose,
although the particular hexose obtained varies in different cases. In fact, the
polysaccharides are often classified according to the hexoses which they
yield on hydrolysis, into the following groups: the dextrosans, which yield
glucose, and include starch, dextrin, glycogen, lichenin, etc.; the levulosans,
which yield fructose, and include inulin, graminin, triticin, etc.; the
mannans; and the galactans. The more common representatives of each of
these groups are discussed below.

(A) The Dextrosans

These are by far the most common type of polysaccharides to be found in
plants.

Starch.—It is probable that no other single organic compound is so
widely distributed in plants as is ordinary starch. It is produced in large
quantities in green leaves as the temporary storage form of photosynthetic
products. As a permanent reserve food material, it occurs in seeds, in fruits,
in tubers, in the pith, medullary rays and cortex of the stems of perennials,
etc. It constitutes from 50 to 65 per cent of the dry weight of seeds of
cereals, and as high as 80 per cent of the dry matter of potato tubers.

Starch occurs in plant tissues in the form of microscopic granules,
composed of concentric layers, there being apparently alternate layers of two
types of carbohydrate material, which have been distinguished from each
other by several different pairs of names used by different authors: thus,
Nägeli uses the terms "granulose" and "amylocellulose"; Meyer, "α and β
amylose"; Wolff, "amylo-cellulose" and "amylo-pectin"; while Kramer

asserts that the layers are alternate lamella of crystalline and colloidal starch.
Many theories as to the nature of these concentric layers and their mode of
deposition have been advanced, but it would not be profitable to discuss
them in detail here.

For purposes of study, starch may be prepared from the ground meal of
cereals, potatoes, etc., by kneading the meal in a bag or sieve of fine-meshed
muslin or silk, under a slow stream of water. The starch granules, being
microscopic in size, readily pass through the cloth with the water, and may
be caught in any suitable container. The starch is then allowed to settle to the
bottom, the water poured off and the starch collected and dried.

Starch is insoluble in water; but if boiled in water, the granules burst and a
slimy opalescent mass, known as "starch paste," is obtained. This is
undoubtedly a colloidal suspension of the starch in water. By various
processes, such as boiling with very dilute acids, treatment with acetone,
etc., starch is converted into "soluble starch" which dissolves in water to a
clear solution. Soluble starch is precipitated out of solution by alcohol, or by
lead subacetate solution.

Air-dried starch contains from 15 to 20 per cent of water; but this can be
completely removed, without altering the starch in any way, by heating for
some time at 100° C.

The starch granules from different sources vary considerably in size and
shape, and can generally be identified by observation under the microscope.

The most characteristic reaction of starch is the blue color which it gives
with iodine. The reaction is most marked with starch paste or soluble starch,
but even dry starch granules are colored blue when moistened with a
solution of iodine in water containing potassium iodide, or with tincture of
iodine.

When hydrolyzed, either by boiling with dilute acids or under the
influence of enzymes, starch undergoes a series of decompositions, yielding
first dextrins, then maltose, and finally glucose. These transformations can
be traced by the iodine color reaction, as starch will show its characteristic
blue, dextrins purple or rose-red, and maltose and glucose no color with
iodine.

Dextrins may occur in plants as transition products in the transformation
of starch into sugars, or vice versa. Most commonly, however, they are
artificial products resulting from the partial hydrolysis of starch in the
laboratory or factory. They are amorphous substances, which are readily
soluble in water, forming sticky solutions which are often used as adhesives
("library paste" is a common example of a very concentrated preparation of
this kind). They are precipitated from solution by alcohol, but not by lead
subacetate (distinction from starch). They are strongly dextrorotatory
(specific rotatory power +192° to +196°); are not fermented by yeast alone,
but readily undergo hydrolysis to glucose which does ferment. There are
several different modifications, or forms, of dextrins, depending upon the
extent to which the simplification of the starch molecule by hydrolysis is
carried. Three fairly definite forms are generally recognized, as follows:
amylo-dextrin, or soluble starch, slightly soluble in cold water, readily so in
hot water, giving a blue color with iodine; erythro-dextrin easily soluble in
water, neutral taste, red color with iodine; and achroo-dextrin, easily soluble
in water, sweetish taste, no color with iodine.

Commercial dextrin, which is much used in the preparation of mucilages
and adhesive pastes, is prepared by heating dry starch to about 250° C. It is
composed chiefly of achroo-dextrin, mixed with varying quantities of
erythro-dextrin and glucose.

Glycogen, or "animal starch," is one of the most widely distributed
reserve foods of the animal body; in fact, it is the only known form of

carbohydrate-reserve in animal tissues. But it is present only rarely in plants.
It occurs in certain fungi, particularly in yeasts. In the animal body, glycogen
is found in all growing cells; also in the muscles and blood; but most largely
in the liver, where it is stored in large quantities. The glycogen found in
yeasts is identical with that found in animal tissues. The quantity of
glycogen in a yeast cell increases rapidly as the yeast grows during the
fermentation process.

Glycogen is a white, amorphous compound, readily soluble in hot water,
forming an opalescent solution similar in appearance to the solutions of
soluble starch. It is strongly dextrorotatory (specific rotatory power +190°),
is colored brown by iodine, and is hydrolyzed to dextrin and maltose, and
finally to glucose.

Lichenin, para dextran, and para isodextran are dextrosans which have
been isolated from various lower plants. They all yield glucose when
completely hydrolyzed. They resemble starch in chemical properties, but
differ from it in physical form, etc.

(B) Levulosans

Inulin replaces starch as the reserve food carbohydrate in a considerable
number of natural orders of plants, particularly in the Compositae. It is the
carbohydrate of the tubers of the dahlia and artichoke and of the fleshy roots
of chicory. It is often found associated with starch in monocotyledonous
plants, such as many species of Iris, Hyacinthus, and Muscari. Among the
monocotyledons, starch seems to be the characteristic carbohydrate reserve
of aquatic, or moisture-loving, species, while inulin is more common among
those which prefer dry situations.

Inulin may be prepared from the tubers of dahlias or artichokes, by boiling
the crushed tubers with water containing a little chalk (to precipitate mineral
salts, albumins, etc.) filtering and cooling the filtrate practically to the
freezing point, which precipitates the inulin.

Inulin is a white, tasteless, semi-crystalline powder, which is soluble in
hot water, from which it may be precipitated by alcohol or by freezing. It
forms no paste like that of starch or dextrin, and gives no color with iodine.
It is levorotatory, and when hydrolyzed by acids or by the enzyme inulinase
yields fructose; in fact, inulin bears the same relation to fructose that starch
does to glucose.

Graminin, irisin, phlein, sinistrin, and triticin are all inulin-like
polysaccharides, which have been found in the plants after which they are
named. Their solutions are, as a rule, sticky or gummy in consistency, which
suggests that these compounds bear the same relation to inulin that dextrins
do to starch.

(C) Mannosans, or Mannans

Mannan bears the same relation to mannose that starch does to glucose
and inulin to fructose. It occurs as a reserve food substance in many plants. It
has been reported as present in moulds, and in ergot; in the roots of
asparagus, chicory, etc.; in the leaves and wood of many trees, such as the
chestnut, apple, mulberry, and many conifers; also as a part of the so-called
"hemi-celluloses" which are present in the seeds of many plants, notably the
palms, the elders, cedar, larch, etc.

It is a white, amorphous powder, which is difficultly soluble in water, is
strongly dextrorotatory (specific rotatory power +285°), and when

hydrolyzed yields mannose.

Secalin (or carubin) is a substance which is found in the seeds of barley,
rye, etc., which is similar to mannan, but is optically inactive.

(D) Galactans

These bear the same relation to galactose that the preceding dextrosans do
to their constituent hexoses. Four different galactans have been isolated from
plant tissues; they are all white, amorphous solids which dissolve with
difficulty in water, forming gummy solutions.

Both galactans and mannans commonly occur associated with cellulose
and hemi-celluloses in the seeds or other storage organs of plants. They are
practically indigestible by animals, as the proper enzymes to hydrolyze them
are not present in the digestive tract; hence, they are commonly classed with
the indigestible cellulose as the "crude fiber" of plants which are to be used
as food by animals.

PHYSIOLOGICAL USE AND BIOLOGICAL
SIGNIFICANCE OF CARBOHYDRATES

If the organic compounds produced by plants be classified with reference
to their uses in metabolism into the three groups known, respectively, as
temporary foods, storage products, and permanent structures, it is clear that
the carbohydrates which have been discussed in this chapter may fall into
either one of the first two of these classes. There can be no doubt that the
first products of photosynthesis, whichever ones they may be in different
plants, may be directly used as temporary foods, to furnish the energy and

material for the building up of permanent structures. Also, there can be no
doubt that these same carbohydrates are translocated to the storage organs
and accumulated for later use by the same plant (as, for example, in the case
of the perennials), or by the next generation of the plant (when the storage is
in the endosperm adjoining the embryo of the seed).

There is no known explanation as to why different species of plants make
use of different carbohydrates for these purposes; or why certain species
elaborate starch out of the same raw materials from which other species
produce sugars, inulin, or glycogen, etc.

In general, starch is the final product of photosynthesis in most green
plants; but there are many exceptions to this. The polysaccharides, which are
generally insoluble, must be broken down into the simpler soluble sugars
before they can be translocated to other organs of the plant for immediate, or
future, use. When they reach the storage organs, they may be recondensed
into insoluble polysaccharides, or stored as soluble sugars. Examples of the
latter type of storage are, sucrose in beet roots, glucose in onion bulbs, etc.
Sometimes, this habit of storage seems to be a species characteristic; as
potatoes store starch, while beets, growing in the same soil and under exactly
the same environment, store sugar. But in other cases, the nature of the
carbohydrate stored undoubtedly is correlated with the external temperatures
at the time of storage. It has been shown that cold, which tends to
physiological dryness, very frequently favors the storage of sugars instead of
starches. Thus, in temperate zones, among aquatic, or moisture-loving
plants, those species which hibernate during the winter at the bottom of lakes
or ponds and are killed by temperatures below freezing, store starch and no
sugar; while in the same ponds, the species whose storage organs pass the
winter above the level of the water and can withstand temperatures as low as
-7° C. contain sugar during the winter months, even if they contain starch
during warmer periods. Similarly, sugars often appear in the leaves and

stems of conifers during the winter months, only to disappear, or be replaced
by starch, when spring approaches. This same phenomenon is noticeable in
arctic plants, which generally contain but small proportions of starch and
relatively large amounts of sugars.

Similarly, the phenomenon of the turning sweet of potatoes when exposed
to low temperatures has often been noted. The change of the starch in potato
tubers to sugar is most rapid at the temperature of 0° C., and ceases at 7°, or
above. Also, if potatoes in which the maximum amount of sugar is present
(not over one-sixth of the total starch can be converted into sugar) are
exposed to a higher temperature the sugar soon disappears.

In general, however, it may be said that each particular species of plant
has its own particular preference for a specific carbohydrate as its reserve
food material, and elaborates the proper enzymes to make it possible to
utilize this particular carbohydrate for its metabolic needs.

Again, the question as to whether the storage of energy-producing
materials for the use of the next generation shall be in the form of
carbohydrates or of fats seems to be definitely connected with the size of the
seed, and the consequent available storage space (see page 138). Animals
habitually use the space-conserving form of fats for their energy-storage,
while plants more commonly use carbohydrates for this purpose, except in
the case of those small seeds in which sufficient energy cannot be stored in
carbohydrate form to develop the young seedling to the point where it can
manufacture its own food. As a general rule, nuts, which contain the embryo
of slow-growing seedlings, and need large proportions of energy reserve, are
characteristically oily instead of starchy in type.

But, aside from temperature reactions and space requirements, there is no
law which has yet been discovered which determines the character of the
energy-storage compound which any given species of plant will elaborate.

The process of photosynthesis would seem to be identical in all cases, at
least up to the point of the production of the first hexose sugar; but the
transformation of glucose into other monosaccharides, disaccharides, and
polysaccharides seems to be a matter which obeys no rule or law.

Finally, there remains to be considered the occurrence and uses of sugars
in the fleshy tissues of fruits. These tissues have, of course, no direct
function in the life history of the plant. They surround the seed, but they
must decay or be destroyed before the seed can come into the proper
environment for germination and growth. In most fruits, starch is the form in
which the carbohydrate material is first deposited in the green tissue, but as
the fruit ripens the starch rapidly changes into sugars, with the result that the
fruit takes on a flavor which makes it much more attractive as a food for
men and animals. This purely biological significance of the presence of
sugars (and of the other substances which give desirable flavors to fruits,
vegetables, etc.), can have no possible relation to the physiological needs of
the individual plant, however.

It is apparent that the production of these immense stores of reserve food
by plants makes them useful as food for animals, and it is, of course, the
storage parts of the plants which are most useful for this purpose. This
biological relationship needs no further emphasis.

REFERENCES

Abderhalden, E.—"Biochemisches Handlexikon, Band 2 ... Die
Einfachen Zuckerarten, Inuline, Cellulosen, ...," 729 pages,
Berlin, 1911, and "Band 8—1 Ergänzungsband (same title as
Band 2)—" 507 pages; Berlin, 1914.

Armstrong, E. F.—"The Simple Carbohydrates and Glucosides,"
233 pages. Monographs on Biochemistry, London, 1919 (3d ed.).

Fischer, E.—"Untersuchung ueber Kohlenhydrate und Fermente,
1884-1908," 912 pages, Berlin, 1909.

Mackensie, J. E.—"The Sugars and their Simple Derivatives,"
242 pages, 17 figs., London, 1913.

Tollens, B.—"Kurzes Handbuch der Kohlenhydrate", 816 pages,
29 figs., Leipzig, 1914 (3d ed.).

CHAPTER V

GUMS, PECTINS, AND CELLULOSES

These substances constitute a group of compounds which are very similar
to the polysaccharide carbohydrates in composition and constitution, but
which serve entirely different purposes in the plant. As a class, they are
condensation products of pentoses, known as pentosans and having the
formula (C5H8O4)n, or hexosans having the formula (C6H10O5)n, or
combined pentosan-hexosans.

In general, these compounds make up the skeleton, or structural
framework material, of the plant, in contrast with the protoplasmic materials
or food substances for which most of the other types of organic compounds
(discussed in other chapters of this book) serve. They are the principal
constituents of "woody fiber," of cell-walls, and of the "middle lamella"
which fills up the spaces between the plant cells. They are, therefore, found
in largest proportions in the stems of woody plants; but they are also present
in every other organ of plants, as the cell-wall or other structural material.

For purposes of study, these compounds may conveniently be divided into
three groups; namely, the natural gums and pentosans, the pectins and
mucilages, and the celluloses. The segregation into these three groups is not
sharply defined. The distinction between the groups is based upon the

solubility of the compounds in water. The gums and pentosans readily
dissolve in water; the pectins form colloidal solutions which are easily
converted into "jellies"; the mucilages do not dissolve but form slimy
masses; while the celluloses are insoluble in and unaltered by water. Some
authors add a fourth group, known as "humins"; but as these are the products
of decay (usually in the soil) of these structural compounds, rather than of
growth and development, they need not be taken into consideration in a
study of the chemistry of plant growth.

THE NATURAL GUMS AND PENTOSANS

The natural gums, when hydrolyzed, yield large proportions of sugars, but
most of them also contain a complex organic acid nucleus, by means of
which they form salts with calcium, magnesium, etc. Some of them, such as
cherry gum and those which are found in the woody stems of plants (wood
gum, and those found in corn stalks, the straw of cereals, etc.) yield
practically pure pentoses. These are known as pentosans. They bear the same
relation to the pentose sugars as do the dextrosans to glucose, etc. The
wound gums, for example, yield arabinose, and the wood gums yield xylose.
But most of the natural gums yield a mixture of galactose, some pentose, and
some complex organic acid.

The gums are translucent, amorphous substances, whose solutions in
water are levorotatory. They are precipitated out of solution by alcohol and
by lead subacetate solution.

Gums are extremely difficult to hydrolyze, the laboratory process of
hydrolysis usually requiring from eighteen to twenty-four hours of
continuous boiling with acids for its completion. Because of this difficulty of

hydrolysis, gums are practically indigestible by animals and of little use as
food.

The following common examples will serve to illustrate the general nature
of these compounds.

Gum arabic, found in the exudate from the stems of various species of
Acacia, is a mixture of the calcium, magnesium, and potassium salts of a
diaraban-tetragalactan-arabic acid. Arabic acid has the formula C23H38O22,
and one molecule of this acid serves as the nucleus for the union of eight
galactose and four arabinose groups, linked together in some unknown way.
The formula for the compound, exclusive of the metallic elements with
which it is loosely united is C91H150O78. This gives some idea of its
complexity.

When boiled with nitric acid, it is oxidized to mucic, saccharic, and oxalic
acids. It gives characteristic reactions with alum, basic lead acetate, and
other common reagents.

Gum arabic comes on the market as a brittle, glassy mass, which is used in
the preparation of mucilages, and as a carrier for essential oils, etc., in
certain toilet preparations.

Recent investigations have shown that the so-called "meta-pectic acid,"
which is often found in sugar beets and interferes with the process of sugar
manufacture, is identical with gum arabic in composition and properties.

Gum tragacanth is the soluble portion of the natural gum which is found
in several species of Astragalus. It constitutes only 8 to 10 per cent of the
total gum-like material which is present, the remainder being composed of
insoluble gummy substances of unknown composition. The soluble gum
consists of calcium, potassium, and magnesium salts of an acid which, when

hydrolyzed, yields several molecules of arabinose, six of galactose, and one
of geddic acid (an isomer of arabic acid). It is said to be produced by the
metamorphosis of the medullary rays under unfavorable conditions of
growth. It comes on the market in globular masses of amorphous material,
and is used in the manufacture of cosmetics, etc.

Wound gum is frequently found in the tracheæ of plants, and near surface
wounds, which it stanches. It is secreted by the cells surrounding the injured
part. It responds to the reactions of other gums and to some of those of
woody fiber. Its exact composition is not known, but probably lies between
that of the true gums and that of cellulose.

These gums are generally considered to be decomposition products of
celluloses, resulting from the action of some hydrolytic ferment, usually
stimulated by some unfavorable condition of growth, some injury, or some
morbid condition.

The pentosans, araban and xylan, occur normally in the stems and outer
seed coats of many common plants. They constitute a considerable
proportion of these tissues, as indicated by the following results of typical
analyses: Wheat bran, 22 to 25 per cent; clover hay, 8 to 10 per cent; oat
straw, 16 to 20 per cent; wheat straw, 26 to 27 per cent; corn bran, 38 to 43
per cent; jute fiber, 13 to 15 per cent; various wood gums, 60 to 92 per cent.

They are white, fluffy solids, which are difficultly soluble in cold water,
more readily in hot water. They are very difficult to hydrolyze, and
indigestible by animals. When finally hydrolyzed, they yield arabinose and
xylose, respectively. The pith of dry corn stalks is a good illustration of their
general character.

MUCILAGES

These are characterized by forming slimy masses when moistened with
water. They are secreted by hairs on the skin of many plants, so that the
external walls of the leaves, fruit, and seeds are often mucilaginous when
damp. This is particularly true of aquatic plants. The chemical composition
of the mucilages is unknown. When hydrolyzed, they yield arabinose and a
hexose; the latter is sometimes galactose and sometimes mannose.

When present on the surface of plant tissues, the mucilages probably serve
to prevent the too rapid diffusion of materials through the skin, in the case of
the aquatic plants, and too rapid transpiration, in the case of young
vegetative tissues or in other plants when growing under extremely dry
conditions. When found in tubers, or other storage organs, it has been
supposed that they may serve as reserve food materials, but it seems that
such difficultly hydrolyzable compounds as these can hardly function as
normal reserve foods.

PECTINS

Many fruits, such as currants, gooseberries, apples, pears, etc., and many
fleshy roots of vegetables, such as carrots, parsnips, etc., contain substances
known as pectins. These are readily soluble in water, and when dissolved in
concentrated solutions in hot water, they set into "jellies" when the solution
is cooled. These jellies carry with them the soluble sugars and flavors which
are present in the fruits, and constitute a familiar article of diet.

There are undoubtedly several different modifications of the pectins, to
which the names "meta-pectin," "para-pectin," "pectic acid," "meta-pectic
acid," and "para-pectic acid," have been applied. These all seem to be
products of hydrolysis of a mother substance known as "pectose," which
constitutes the middle lamella of unripe fruit, etc. As the fruit ripens, the

pectose is hydrolyzed into the various semi-acid, or acid, bodies mentioned
above. The intermediate products of the hydrolysis are the pectins, which
swell up in water and readily form jellies; while the final meta-pectic acid is
easily soluble in water and resembles the true gums in its properties. When
the middle lamella reaches the pectic acid stage, the fruit becomes soft and
"mushy" in texture.

The pectins more nearly approach to the composition, properties, and
functions of the celluloses than do any of the other groups of organic
compounds. They have been extensively studied in connection with the
parasitism of certain fungous diseases which cause the soft rots of fruits and
vegetables. These parasites usually penetrate the tissues of the host plant by
dissolving out the middle lamella material, which may sometimes serve as
food material for the fungus; but more often the parasite secures its food
supply from the protoplasm of the cell contents. In such cases, the parasite
secretes both a pectose-dissolving enzyme, known as "pectase" and a
"cellulase" which attacks the cell-wall material in order to provide for the
entry of the fungus into the cells. Other enzymes, known as "pectinases,"
which coagulate the soluble pectins or pectic acids into insoluble jellies in
the tissues of the plants seem to aid the plant in resisting the penetration by
the parasite.

CELLULOSES

Used in its general sense, this term includes all those substances which are
elaborated by protoplasm to constitute the cell-wall material. Cellulose
proper is a definite chemical compound, whose properties are well
established. In plants, however, this true cellulose is nearly always
contaminated by various encrusting materials; and in the process of wood-
formation, the cell-wall material continually thickens by the conversion of

the cellulose into ligno-cellulose and the protoplasm of the cell as
continuously diminishes in volume. Thus the protoplasm of the cell produces
a number of different kinds of material which are deposited in the walls of
the cell. All of these, taken together, constitute the general group known as
the celluloses.

These may be divided into three classes: namely, (1) the hemi-celluloses,
(2) the normal celluloses, and (3) the compound celluloses.

The hemi-celluloses (pseudo-, or reserve celluloses) include a series of
complex polysaccharides which occur in the cell-walls of the seeds of
various plants. They are found in the shells of nuts, rinds of cocoanuts, shells
of stony fruits, etc., and in the seedcoats of beans, peas and other legumes.
They are much more easily hydrolyzed than the other members of this
group, and when hydrolyzed yield various sugars, chiefly galactose,
mannose, and the pentoses. They bear the same relation to these sugars that
starch does to glucose, and are generally supposed to serve as reserve food
material, although it is difficult to conceive how the shells, etc., in which
they appear can be utilized by a growing seedling. They differ in structure
from the fibrous celluloses and are probably not cell-wall building material.
They appear to be a form of reserve carbohydrates, which differ from the
glucose-polysaccharides in being condensed in, or as a part of, the external
structural material rather than in the internal storage organs. They are soluble
in water and exhibit the properties of gums, and are often classified with the
gums and described under the names "galactans," "mannosans," "pentosans,"
etc.

The normal celluloses, of which the fibers obtained from cotton, flax,
hemp, etc., are typical examples, are widely distributed in plants and form
the commercial sources for all textile fibers of vegetable origin. Ordinary
cotton fiber contains 91 per cent of cellulose, about 7.5 per cent of water, 0.4
per cent of wax and fat, 0.55 per cent of pectose derivatives, and 0.25 per

cent of mineral matter; or a total of only 1.2 per cent of non-cellulose solids.
Filter paper is practically pure cellulose.

Pure cellulose is a white, hygroscopic substance, which is insoluble in
water and in most other solvents. If heated with water under pressure to
about 260° C., it dissolves completely without decomposition. If boiled with
a strong solution of zinc chloride, or treated in the cold with zinc chloride
and concentrated hydrochloric acid, or with an ammoniacal solution of
copper hydroxide (Schweitzer's reagent), it dissolves to a clear solution from
which it may be reprecipitated without chemical change by neutralizing or
diluting the solution.

Cellulose has the formula (C6H12O5)n. When hydrolyzed under the
influence of the enzyme cytase, it breaks down, first into cellobiose, an
isomer of maltose, and then into glucose. It is, therefore, chemically like, but
not identical with, starch; and structurally it is arranged in fibrous form
instead of in granules. Under the action of fermentative enzymes, as when
vegetable matter decays under stagnant water, in swamps, etc., cellulose
breaks down into carbon dioxide and marsh gas, according to the equation

(C6H12O5)n + nH2O = 3nCO2+3nCH4.

Cellulose is acted upon by caustic alkalies in a variety of ways. When
fused with a mixture of dry sodium and potassium hydroxides, it is
decomposed into oxalic and acetic acids. When heated with a 10 to 15 per
cent solution of caustic soda, cellulose fibers thicken and become
translucent, thus resembling silk fibers. This process, known as
"Mercerizing," is largely used for the production of commercial fabrics.

Acids also act on cellulose in a variety of ways. When heated with nitric
acid (sp. gr. 1.25), it is converted into oxycellulose; while dilute sulfuric
acid, under similar conditions, yields hydro-cellulose, a substance having the

formula C12H22O11, which retains the fibrous structure of the original
cellulose but which, when dry, may be rubbed up into a fine powder.
Concentrated nitric acid, or better, a mixture of concentrated nitric and
sulfuric acids, acts upon cellulose, converting it into various nitro-
derivatives, several of which have great industrial value. The number of NO3
groups which unite with the cellulose molecule under these conditions
depends upon the temperature, pressure, etc., employed during the nitration
process; di-, tri-, tetra-, penta-, and hexanitrates are all known. Pyroxylin, or
collodion, is a mixture of the tetra- and penta-nitrates, which is soluble in
alcohol and is used in surgery, in photography, and in the manufacture of
celluloid, which is a mixture of collodion and camphor. The hexanitrate,
C12H14(NO3)6O4, is the violent explosive known as gun-cotton.

Gentler oxidizing agents, such as "bleaching powder," etc., have no effect
upon cellulose, and hence are extensively used in the treatment of cotton and
other vegetable fibers, in preparation for their use in the manufacture of
textiles, paper, etc.

Cellulose is indigestible in the alimentary tract of animals, but the
putrefactive bacteria which are generally present there ferment it, with the
production of acids of the "fatty acid" series, carbon dioxide, methane, and
hydrogen. Excessive fermentations of this kind are responsible for the
distressing phenomenon known as "bloat."

The compound celluloses comprise the larger proportion of the material
of the woody stems of plants. They consist of a base of true cellulose, which
is either encrusted with or chemically combined with some non-cellulose
constituent. Depending upon the nature of the non-cellulose component, the
compound celluloses are divided into three main groups, known respectively
as (1) ligno-celluloses, (2) pecto-celluloses, and (3) adipo-, or cuto-
celluloses. As the names indicate, the non-cellulose component in the first

group is lignin; in the second, pectic substances; and in the third, fats or
waxes.

Ligno-celluloses.—In the young plant cell, the cell-walls consist of
practically pure cellulose; but as the plant grows older, this becomes
permeated with lignin, or woody fiber, until in the stem of a tree, for
example, the proportion of cellulose in the tissue is only 50 to 60 per cent. In
the preparation of wood pulp for the manufacture of paper, the lignin
materials are dissolved off by means of various chemical reagents, leaving
the cellulose fibers in nearly pure form for use as paper. The lignin material
generally consists of two types of substances, one of which contains a
closed-ring nucleus of unknown composition and the other is probably a
pentosan. These materials are so extremely difficult to hydrolyze that their
composition has not yet been definitely determined.

Pecto-celluloses are found in various species of flowering plants; those
which are present in the stems and roots being true pecto-celluloses, while
those which are found in fruits and seeds contain mucilages rather than
pectose derivatives, and are generally designated as "muco-celluloses." The
exceedingly inert character of these compounds makes their study difficult
and their functions uncertain.

The term cuto-celluloses is applied to the group of substances, including
suberin and cutin, which constitute waterproof cell-walls. These were
formerly supposed to consist of true cellulose impregnated with fatty or
wax-like materials. Recent investigations seem to indicate, however, that
there is really no cellulose nucleus in such walls as these, but that they are
compound glyceryl esters resembling the true fats (see Chapter X) in
composition. If this view should finally be established as a fact, this sub-
group of supposed compound celluloses should be dropped from
consideration as such.

PHYSIOLOGICAL USE OF CELLULOSES

There seems to be no question that the sole use of celluloses is to serve as
structure-building materials. They are undoubtedly elaborated from the
carbohydrates as the cell grows. In only rare cases, however, is there any
evidence that they can be reconverted into carbohydrates to serve as food
material. Certain bacteria can make use of cellulose as food, and secrete an
enzyme, cytase, which aids in the hydrolysis of cellulose to sugars for this
purpose. But this enzyme seems rarely, if at all, to be present in the tissues of
higher plants. It has been reported that some cellulose is hydrolyzed during
the malting of barley, indicating that this might have some food use for the
growing seedling; but this observation has not been confirmed and later
investigations seem to throw doubt upon its accuracy.

Bacteria of decay also act upon cellulose materials, converting them
chiefly into gaseous products; but this seems to be a provision of nature for
the destruction of the cell-wall material of dead plants, rather than an
arrangement for the constructive use of it as food for the bacterium. When
fibrous plant residues decay in the soil, the cellulose compounds are first
converted into a series of complex organic acids, known as "humins," which
undoubtedly have a significant effect upon the chemical and physical
properties of the soil, but these have little interest or significance in a study
of the chemistry of plant growth.

REFERENCES

Abderhalden, E.—"Biochemisches Handlexikon, Band 2,
Gummisubstanzen, Hemicellulosen, Pflanzenschleimen ..." 729
pages, Berlin, 1911; and "Band 8—1 Ergänzungsband (same title
as Band 2)—," 507 pages, Berlin, 1914.

Schwalbe, C. G.—"Die Chemie der Cellulose," 665 pages,
Berlin, 1911.

CHAPTER VI

GLUCOSIDES

Strictly speaking, the term glucoside should be applied only to such
compounds as contain glucose as the characteristic basic group. But in
common usage, it refers to any compound which, when hydrolyzed, yields a
sugar as one of the products of the hydrolysis. In all the natural glucosides
which occur in plant tissues, the other organic constituent, which is
represented by the R in the formula for glucosides (R·C6H11O5, or
R·(CHOH)5CHO) is some aromatic group, or closed-ring benzene
derivative.[3] The different organic constituents of glucosides are of a great
variety of types, such as phenols, alcohols, aldehydes, acids, oxyflavone
derivatives, mustard oils, etc. It is noteworthy, however, that no nitrogenous
groups of the protein type have been found combined with sugars in
glucosides.

Some glucosides contain more than one saccharide group, possibly as di-
or trisaccharides. Under proper conditions of hydrolysis, one or more of the
saccharide groups can be removed from such compounds, resulting in
glucosides of simpler structure.

Most of the common glucosides are derived from d-glucose. Some are
known, however, which are derivatives of galactose or rhamnose; while in
some cases the exact nature of the sugar which is present has not yet been
determined.

FOOTNOTES:

[3
]

The structural formula for benzene, C6H6, is one which it is
difficult and inconvenient to reproduce in type. On that account, it is customary to indicate this

formula by a plane hexagon, thus .

It is understood, in all such cases, that the figure represents six carbon atoms arranged in a
closed ring, with alternate double and single bonds, and with a hydrogen atom attached to each
carbon. The printing of some other group as OH, CH3, adjacent to an angle of the hexagon
means that this group replaces the H atom in the compound which is being illustrated.

HYDROLYSIS OF THE NATURAL GLUCOSIDES

All natural glucosides are hydrolyzed into a sugar and another organic
residue by boiling with mineral acids; although they vary widely in the ease
with which this hydrolysis is brought about.

In most cases, the glucoside is easily hydrolyzed by an enzyme which
occurs in the same plant tissue, but in different cells than those which
contain the glucoside. Injury to the tissues, germination processes, and
perhaps other physiological activities of the cells, result in bringing the
enzyme in contact with the glucoside and the hydrolysis of the latter takes
place. A large number of such enzymes have been found in plants, many of
which hydrolyze only a single glucoside. However, two enzymes, namely,
the emulsin of almond kernels, and myrosin of black mustard seeds, each
hydrolyze a considerable number of glucosides. In general, emulsin will aid
in the hydrolysis of any glucoside which is a derivative of β-glucose, and
myrosin will help to split up any sulfur-containing glucoside. Glucosides
which are derivatives of rhamnose require a special enzyme, known as
rhamnase, for their hydrolysis.

The following reactions for the hydrolysis of arbutin and of amygdalin are
typical of this action, and will serve to illustrate the general structure of these
compounds:

GENERAL PROPERTIES OF GLUCOSIDES

As a rule, glucosides are easily soluble in water. They are generally
extracted from plant tissues by digestion with water or alcohol. In most
cases, the enzyme which is present in other cells of the same tissue must be
killed by heating the material, in a moist condition, to the temperature of
boiling water, before the extraction is begun, as otherwise the glucoside will
be hydrolyzed as rapidly as it is extracted from its parent cell. Maceration or
otherwise bruising the tissue, after the enzyme has been destroyed, facilitates

the extraction. The glucosides, after extraction and purification by
recrystallization, are generally colorless, crystalline solids, having a bitter
taste and levorotatory optical activity. This latter property is remarkable, as
most of them are compounds of the strongly dextrorotatory d-glucose.

Many of the natural glucosides have marked therapeutic properties and are
largely used as medicines; others are the mother-substances for brilliant
dyes; for example, indican, from which indigo is obtained, and the alizarin
glucosides.

Several hundred different glucosides have been isolated from plant
tissues, and their properties described, and this number is being added to
constantly, as the methods of isolation and study are improved. They may be
classified into groups, according to the nature of the organic compound other
than sugars which they yield when hydrolyzed. The following descriptions
of the occurrence, constitution, products of hydrolysis, and special properties
of typical members of each of the several different classes of glucosides will
serve to illustrate their general relationship to plant growth.

THE PHENOL GLUCOSIDES

Arbutin, C12H16O7, is obtained from the leaves of the bear berry
(Arctostaphylos uva-ursi), a small evergreen shrub. When hydrolyzed by
mineral acids or emulsin, it yields glucose and hydroquinone.

C12H16O7 + H2O = C6H12O6 + C6H4(OH)2.

Hydroquinone has strongly antiseptic properties. Arbutin is both an
antiseptic and a diuretic, and is used in medicine.

Phloridzin, C21H24O10, is found in the bark of apple, pear, cherry, plum,
and similar trees. Mineral acids (but not emulsin) hydrolyze it to glucose and
phloretin (C15H14O5), according to the equation

It is used in medicine as a remedy for malaria, having marked anti-
periodic properties.

Glycyphyllin, C21H24O9, found in leaves of Smilax, yields rhamnose and
phloretin, when hydrolyzed.

Iridin, C24H26O13 (glucose and irigenin), found in rootstocks of Iris, is
used in medicine as a cathartic and diuretic.

Baptisin, C26H32O14·9H2O (two rhamnose and baptigenin), found in
roots of wild indigo (Baptisia), has strong purgative properties.

Hesperidin, C50H60O27 (one rhamnose + two glucose + hesperitin), is
found in the pulp of lemons and oranges.

The characteristic phenol group which is present in these glucosides has
the following structural formula, in each case, the X indicating the H atom
which is replaced by the sugar molecule to form the glucoside:

THE ALCOHOL GLUCOSIDES

Salicin, C13H18O7 (glucose + saligenin, or o-oxy benzyl alcohol) is found
in the bark, leaves, and flowers of most species of willow, the proportion
present depending upon the season of the year, and the sex of the tree. It is
used as a remedy against fevers and rheumatism, causing less digestive
disturbances than the salicylic acid which is the oxidation product of
saligenin and which is sometimes used as a remedy for rheumatism.

Coniferin, C16H22O8 (glucose and coniferyl alcohol), is found in the bark
of fir trees. The coniferyl alcohol obtained from coniferin by hydrolysis can
be easily oxidized to vanillin, and is, therefore, the source for the artificial
flavoring extract used as a substitute for the true extract of the vanilla bean.

Populin, C20H22O8 (glucose + saligenin+benzoic acid), found in the bark
of poplar trees, is used in medicine as an antipyretic. It can be hydrolyzed,
by a special enzyme, into salicin and benzoic acid.

The structure of the two typical closed-ring alcohols which are present in
these glucosides is indicated by the following formulas;

THE ALDEHYDE GLUCOSIDES

Salinigrin, C13H16O7 (glucose and m-oxy benzaldehyde), is found in the
bark of one species of willow (Salix discolor). Its isomer, known as helicin
(glucose and o-oxy benzaldehyde, or salicylic aldehyde), does not occur
naturally in any plant, but is easily produced artificially by the gentle
oxidation of salicin. Their relationships are shown on the following
formulas;

Amygdalin, also contains a benzaldehyde group, but there is linked with
it a hydrocyanic acid group; hence, this glucoside is usually classed with the
cyanophoric glucosides (see page 86).

THE ACID GLUCOSIDES

The most common example of this group is gaultherin, C14H18O8, which
is found in the bark of the black birch and is a combination of glucose with
methyl salicylate. Both the glucoside itself and the methyl salicylate ("oil of
wintergreen") which is derived from it are used as remedies for rheumatism.

Jalapin, C44H56O16 (glucose and jalapinic acid), and convolvulin,
C54H96O27 (glucose + rhodeose + convolvulinic acid), are glucosides of very

complex organic acids, found in jalap resin, which are used in medicine as
cathartics or purgatives.

THE OXY-CUMARIN GLUCOSIDES

Cumarin itself is widely distributed in plants. No glucoside containing
cumarin as such has yet been isolated; but several glucosides of its oxy-
derivatives are known. The following are common ones:

Skimmin, C15H16O8 (glucose and skimmetin), is found in Skimmia
japonica; æsculin, C15H16O9 (glucose and æsculetin), is found in the bark of
the horse-chestnut, Æsculus hippocastanum, and its isomer, daphnin
(glucose and daphnetin), in several species of Daphne; and fraxin,
C16H18O10 (glucose and fraxetin), is found in the bark of several species of
ash.

The structural arrangement of the oxy-cumarin groups which are found in
these glucosides is shown in the following formulas. It is not known to
which OH group the sugar is attached, in each case.

Scopolin, C22H28O14, found in Scopolia japonica, contains two glucose
molecules united to a monomethyl ether of æsculin; while limettin, found in
certain citrus trees, is the dimethyl ether of æsculin.

THE PIGMENT GLUCOSIDES

Many, if not all, of the red, yellow, violet, and blue pigments of plants
either exist as, or are derived from, glucosides. These are of three types: the
madder, or alizarin, reds are derivatives of various oxy-anthraquinones; most
of the soluble yellow pigments are glucosides derived from flavones or
xanthones; and the soluble red, blue, and violet pigments of the cell-sap of
plants are mostly anthocyan derivatives. The four basic groups, or nuclei,

which are present in these different types of compounds are complex groups
consisting essentially of two benzene rings linked together through a third
ring in which there are either two oxygen atoms in the ring, or one oxygen in
the ring and a second attached to the opposite carbon in the (C=O)
arrangement, as shown by the following diagrammatic formulas:

The red dyes which were formerly obtained from madder, the powdered
roots of Rubia tinctoria, but are now almost wholly artificially synthetized,
consist of at least four different glucosides, the organic group of which, in
each case, is an hydroxy-derivative of anthraquinone. The most important of
these is ruberythric acid, composed of two molecules of glucose linked with
one of alizarin (1,2, dioxyanthraquinone). Xanthopurpurin contains 1,3,
dioxyanthraquinone, which is isomeric with alizarin; and rubiadin is a

monomethyl (the CH3 being in the 4 position), derivative of this compound.
Purpurin is a glucoside of 1,2,4, trioxyanthraquinone.

The soluble yellow pigments are generally glucosides of hydroxy-
derivatives of xanthone or flavone, known as oxyxanthones or oxyflavones.
The sugars which are united to these nuclei vary greatly, so that there are a
great variety of yellow, white, or colorless flavone or xanthone pigment
compounds. These compounds are almost universally present in plants. For
example, one typical set of examinations of the wood, bark, leaves, and
flowers of over 240 different species of tropical plants showed that flavone
derivatives were present in every sample which was tested, the pigments
being usually located in the powdery coating of the epidermis of the tissues.

The following typical examples will serve to illustrate the composition
and properties of the glucosides of this type.

Quercitrin, C21H20O11, is found in oak bark, in the leaves of horse-
chestnut, and in many other plants, often associated with other pigments. It
is a brilliant yellow crystalline powder. Industrially, it ranks next to indigo
and alizarin in importance as a natural dye stuff. It is a glucoside of
rhamnose with 1,3,3',4', tetraoxyflavonol (i.e., the flavone nucleus with five
OH groups replacing the hydrogens in the 1, 3, 5, 3', and 4' positions).
Quercetin, C15H10O7, which is the tetraoxyflavonol itself, without any sugar
in combination with it, is found in the leaves of several species of tropical
plants and in the bark of others. Isoquercitrin, C21H20O12, is derived from
the same flavone, but contains glucose instead of rhamnose, as the sugar
constituent of the glucoside.

Apiin, C26H20O9, the yellow glucoside found in the leaves of parsley,
celery, etc., contains apiose (a pentose sugar of very unusual structure,
represented by the formula,

and apigenin, which is a 1,3,4', trioxyflavone.

Xanthorhamnin, C34H42O20, is a very complex glucoside containing two
rhamnose and one galactose groups, united with rhamnetin, which is
quercitin with the H of the OH in either the 1, or 3, position replaced by a
methyl group. There are several similar pigments which differ from
xanthorhamnin only in the number or position of the methoxy groups (i.e.,
the OH groups with a CH3 replacing the H), or in the nature of the sugar
which is present in the compound. Rhamnetin itself is found in the fruits of
certain species of Rhamnus, and is used in dyeing cotton.

The structural arrangement of the characteristic groups of these flavone
pigments will be dealt with more in detail in the chapter dealing with
Pigments (Chapter VIII).

The best-known yellow pigment which is a xanthone derivative is
euxanthic acid, known as "Indian yellow," which is a "paired" compound of
glucuronic acid (see page 42) and euxanthone. The latter is a 2, 3',
dioxyxanthone. The pigment is found in the urine of cattle which have been
fed on mango leaves.

The soluble red, blue, and violet pigments are glucosides of various
hydroxy-derivatives of the anthocyan nucleus. Their constitution and
properties will be discussed in detail in the chapter dealing with the
Pigments. These compounds are isomeric with similar flavone and xanthone
derivatives, and the transition from one color to the other in plants takes

place very easily under the action of oxidizing or reducing enzymes. This
accounts for the change of reds and blues to yellows and browns, and vice
versa, under changing temperature conditions.

The following red or blue plant pigments, which are anthocyan
glucosides, have been isolated and studied (for the structural arrangement of
the characteristic groups, see pages 116): from cornflower and roses, cyanin,
C28H31O16Cl (2 molecules glucose + cyanidin); from cranberries, idain,
C21H21O10Cl (galactose + cyanidin); from geranium, pelargonin,
C27H30O15Cl (2 molecules glucose + pelargonidin); from pæony, pæonin,
C28H33O16Cl (2 molecules glucose + pæonidin, a monomethyl cyanidin);
from blue grapes, œnin, C23H25O12Cl (glucose + œnidin); from whortle
berry, myrtillin, C22H23O12Cl (glucose + myrtillidin); from larkspur,
delphinin, C41H39O21Cl (2 molecules glucose + 2 molecules p-oxybenzoic
acid + delphinidin); and from mallow, malvin, C29H35O17Cl (2 molecules
glucose + malvidin).

The blue dye, indigo, is derived from a glucoside of an entirely different
type, known as indican. Indican is readily extracted from the leaves of
various species of indigo plants. When hydrolyzed, it yields glucose and
indoxyl (colorless). Indoxyl is easily oxidized to indigotin (the deep blue dye
known as "indigo"). The equations illustrating these changes are as follows:

(a) C14H17O6N + H2O = C6H12O6 + C8H7ON
Indican Glucose Indoxyl
(b) 2C8H7ON + O2 = C16H10O2N + 2H2O
Indoxyl Indigotin

The structural relationships of indoxyl and indigotin may be illustrated by
the following formulas:

Natural indigo dye is prepared by fermentation of indigo leaves, the decay
of the cell-walls liberating the enzymes in the tissues, which bring about the
chemical changes illustrated in the above equations.

THE CYANOPHORE GLUCOSIDES

Several glucosides which yield hydrocyanic acid as one of the products of
their hydrolysis are of common occurrence in plants. These are generally
spoken of as the "cyanogenetic" glucosides; but as they do not actually
produce cyanogen compounds, but only liberate them when hydrolyzed, the
recently suggested term "cyanophore" undoubtedly more correctly indicates
their properties.

The best known and most widely distributed of these is amygdalin.
Amygdalin was first discovered in 1830, and was one of the first substances
to be recognized as a glucoside. It is found in large quantities in bitter
almonds and in the kernels of apricots, peaches, and plums; also in the seeds
of apples, etc., in fact in practically all the seeds of plants of the Rose family.
It is the mother substance for "oil of bitter almonds," which is widely used as
a flavoring extract.

Amygdalin has been the object of very extensive studies, and even yet the
exact nature of the linkage between its constituent groups is not certainly
known. When completely hydrolyzed, it yields two molecules of glucose and
one each of benzaldehyde and hydrocyanic acid. Recent studies indicate that
the two sugar molecules are separately united to the other constituents, rather
than united with each other in the disaccharide relationship. In other words,
amygdalin is a true glucoside rather than a maltoside. This is indicated by
the fact that when submitted to the action of all known hydrolyzing agents
which affect it, it has never been found to yield maltose as one of the
products of hydrolysis. Furthermore, the rate of hydrolysis of amygdalin is
not affected by the presence of maltose; and the segregation of the two
glucose molecules is accomplished by enzymes other than maltase, which is
the only enzyme which is known to break up a maltose molecule. Since the
exact nature of the linkage is not known, it is customary and convenient to
indicate the unit groups as linked together in the following order:

C6H11O5—O—C6H10O4—O—C6H5·CH —C ≡N
(1) (2) (3) (4)

A study of the hydrolysis reactions of amygdalin shows that there are
three different linkages in the molecule which may be broken by the simple
interpolation of a single molecule of water and a fourth which may be split
by a different type of hydrolysis, namely, the C≡N linkage. These are
indicated by the numbers below the corresponding portion of the formula
above. Most hydrolyzing agents break the molecule first at (1), yielding one
molecule of glucose and one of mandelo nitrile glucoside (see page 77). The
next step usually breaks the latter at the point indicated by (2), yielding
glucose and benzaldehyde cyanhydrin, or mandelo nitrile. The latter in turn
breaks down at (3) into benzaldehyde and HCN. But when amygdalin is
boiled with concentrated hydrochloric acid, the first change is the splitting
off at (4) of the nitrogen in the form of ammonia and the consequent

conversion of the CN group into a COOH group, producing amygdalinic
acid. On further hydrolysis, this breaks up in the same order as before.
Similarly, it is possible to convert mandelo nitrile into mandelic acid by
splitting off the nitrogen to form a COOH group, instead of splitting off the
HCN group leaving benzaldehyde.

The mandelo nitrile glucoside contains an asymmetric carbon atom which
is wholly outside its glucose group, thus C6H10O5—O—C6H5·CH·CN.
Hence, it may exist in dextro, levo, and racemic forms. In the amygdalin
molecule, it exists in the dextro form, which has been named "prunasin." The
levo form, known as "sambunigrin," has been obtained by hydrolysis of a
compound isomeric with amygdalin, whose composition has not been
definitely worked out; while the racemic form, known as "prulaurasin," has
been prepared from isoamygdalin, by the action of alkalies. Hence, all the
possible compounds indicated by the presence of the asymmetric carbon
have been found and identified.

The crude enzyme preparation which is obtained from almond seeds,
known as "emulsin," contains two enzymes, amygdalase, which breaks the
amygdalin molecule at linkage (1), and prunase, which breaks it at (2). The
action of amygdalase must always precede that of prunase. In other words, it
is never possible to break off a disaccharide sugar from the molecule, either
by the action of prunase alone, or by means of any other hydrolytic agent.

Dhurrin, C14H17O7N, is another glucoside of fairly general occurrence in
plants, which yields HCN as one of the products of its hydrolysis. It is found
in the leaves and stems of several species of millets and sorghums. Frequent
cases of poisoning of cattle from eating of these plants as forage have been
reported. On hydrolysis, dhurrin first yields glucose and paraoxy-mandelo
nitrile; the latter then breaks down into paraoxy-benzaldehyde and HCN.

Vicianin, C19H25O10N, is a cyanophoric glucoside, found in the seeds of
wild vetch, etc. On hydrolysis, it yields glucose, arabinose, and d-mandelo
nitrile. It is, therefore, similar to amygdalin, except that one glucose
molecule is replaced by arabinose.

THE MUSTARD OIL GLUCOSIDES

The seeds of several species of plants of the Cruciferæ or mustard family
contain glucosides in which the other characteristic group is a sulfur-
containing compound. These glucosides yield "mustard oils" when they are
hydrolyzed by the enzyme myrosin, which accompanies them in the plant.
The following glucosides, found in the seeds of white and black mustard, are
the best-known representatives of this class.

Sinigrin, C10H16O9NS2K, found in black mustard seeds, when
hydrolyzed yields glucose, acid potassium sulfate, and allyl isosulfocyanide
(mustard oil), as indicated by the equation.

C10H16O9NS2K + H2O = C6H12O6 + C3H5≡N=C=S+KHSO4.

The acid potassium sulfate group separates first and most readily, leaving
a compound known as merosinigrin, for which the following formula has
been suggested:

This compound usually breaks down into glucose and mustard oil; but by
special treatment it is possible to obtain from it thioglucose, C6H11O5·SH.
This indicates that in the original glucoside the glucose is linked with the
mustard oil through the sulfur atom.

Sinalbin, C30H42O15N2S2, from white mustard seeds, when hydrolyzed
by myrosin, yields glucose, sinalbin mustard oil (a paraoxybenzyl derivative
of allyl isosulfocyanide) and sinapin acid sulfate; according to the equation

C30H42O15N2S2 + H2O = C6H12O6 + C7H7O·NCS + C16H24O5N·HSO4.
Sinalbin mustard Sinapin acid
Sinalbin Glucose
oil sulfate

The sinalbin mustard oil may be represented by the formula

. Hydrolysis of the sinapin acid sulfate converts it into
sinapinic acid, C6H2OH·(OCH3)2·CH=CH·COOH, choline,
N(CH3)4C2H4OH (see page 152), and H2SO4. It is, therefore, a very
complex glucoside.

TEE DIGITALIS GLUCOSIDES

The five, or more, glucosides which are present in the leaves and seeds of
the foxglove (Digitalis purpurea) have been extensively studied, as they are
the active principles in the various digitalis extracts which are used in
medicine as a heart stimulant.

Digitoxin, C34H54O11, which is the most active of these glucosides in its
physiological effects, when hydrolyzed, yields digitoxigenin, C22H32O4, and
a sugar having the formula C6H12O4, which is known as "digitoxose" and is
supposed to be a dimethyl tetrose.

Digitalin, C35H56O14, is also strongly active. When hydrolyzed, it yields
digitaligenin, C22H10O3, glucose, and digitoxose.

Digitonin, C54H92O28, constitutes about one-half of the total glucosides in
the extract which is obtained from most species of the digitalis plants. It is
much less active than the others. It is a saponin (see page 90) in type. On
hydrolysis, it yields 2 molecules of glucose, 2 of galactose, and one of
digitogenin.

Gitonin, C49H80O23, containing 3 molecules of galactose, one of a
pentose sugar, and one of gitogenin; and gitalin, C28H48O10, containing
digitoxose and gitaligenin, have also been isolated from digitalis extracts.

The structural arrangement of the characteristic groups in these glucosides
has not yet been definitely worked out.

Cymarin, the active principle of Indian hemp (Apocynum cannabinum), is
similar in type to the digitalis glucosides. When hydrolyzed, it yields a sugar
known as "cymarose," C7H14O7, which seems to be a monomethyl
derivative of digitoxose, and cymarigenin, C23H30O5, a compound which is
either identical or isomeric with the organic residue obtained from other
members of this group.

THE SAPONINS

The saponins constitute a group of glucosides which are widely
distributed in plants, whose properties have been known since early Grecian
times. They have been found in over four hundred different species of plants,
belonging to more than forty different orders.

The most characteristic property of saponins is that they form colloidal
solutions in water which produce a soapy foam when agitated, and are
peculiarly toxic, especially to frogs and fishes. In dry form, they have a very
bitter, acrid taste, and their dust is very irritating to the mucous membranes
of the eye, nose, and throat.

On hydrolysis, the saponins yield a variety of sugars,—glucose, galactose,
arabinose, and sometimes fructose, and even other pentoses—and a group of
physiologically active substances, known as "sapogenins."

The more toxic forms of these glucosides are known as "sapotoxins."

The chemical composition of the saponins varies so widely that it is
scarcely possible to cite typical individuals. Sarsaparilla, the dried root of
smilax plants, contains a mixture of non-poisonous saponins, from which at
least four individual glucosides have been isolated and studied. Corn cockle
contains a highly poisonous sapotoxin which, on hydrolysis, yields four
molecules of a sugar and one of sapogenin, C10H16O2. Other sapotoxins are
obtained from the roots of soapwort and from several species of Gypsophila.
Digitonin and digito-saponin are glucosides of this type which are found in
the extracts from various species of Digitalis.

THE PHYSIOLOGICAL USES OF GLUCOSIDES

It is scarcely conceivable that substances which vary so widely in
composition as do the different types of glucosides can possibly all have
similar physiological uses in plants. The cyanophoric glucosides, the
pigment glucosides, the mustard oil glucosides, and the saponins, for
example, can hardly be assumed to have the same definite relationships to
the metabolism and growth of the plant. To be sure, they are alike in that
they all contain one or more sugar molecules, and it is probable that the
carbohydrates which are held in this form may serve as reserve food
material, especially when the glucoside is stored in the seeds; but it is
obvious that the simpler and more normal form of such stored food is that of
the polysaccharides which contain no other groups than those of the
carbohydrates. It seems much more probable that the physiological uses of
glucosides depend upon their ability to form temporarily inactive "pairs"
with a great variety of different types of organic compounds which are
elaborated by plants for a variety of purposes.

It has been noted that in most, if not all, instances, the glucosides are
accompanied in the same plant tissue (although in separate cells) by the
appropriate enzyme to bring about their hydrolysis and so set free both the
sugar and the other characteristic component whenever the conditions are
such as to permit the enzyme to come in contact with the glucoside. This
occurs whenever the tissue is injured by wound or disease, and also during
the germination process.

Injury to the plant tissue seems to be a necessary preliminary to the
functioning of the active components of the glucoside, except in the case of
the seeds. This leads naturally to the supposition that at least some of these
glucosides are protective or curative agents in the plant tissues. This
conception is further supported by the facts that many of the non-sugar
components of glucosides are bactericidal in character and that the
glucosides commonly occur in parts of the plant organism which are

otherwise best suited to serve as media for the growth of bacteria. Thus, it is
known that in the almond, as soon as the tissue is punctured, amygdalin is
hydrolyzed and all bacterial action is inhibited. Similarly, the almost
universal presence of glucosides containing bactericidal constituents in the
bark of trees insures natural antiseptic conditions for all wounds of the outer
surfaces of the stem of the plant. In fact, it is easily conceivable that at least
one of the reasons for the failure of the processes of decay of plant tissues to
set in until after the death of the cells, is that during living, respiratory
activity these antiseptic glucosides are so generally present in the tissues.

Further, it has been fairly well established that the "chromogens," or
mother-substances of the pigments, which, under the influence of oxidase
enzymes, serve to regulate the respiratory activities of the plant are
essentially glucosidic in character. This, and other, functions of the pigments,
most of which are glucosides, will be discussed at some length in the chapter
dealing with the Pigments (Chapter VIII).

Many gaseous anæsthetics are known to have a marked effect in
stimulating plant growth. In a number of cases, it has been shown that the
contact of plant tissues with these anæsthetics brings about an interaction of
the enzyme and glucoside which are present in the tissue, with the
consequent hydrolysis of the latter, setting free its characteristic components.
This observation has led to the supposition that many of the organic
constituents of glucosides are definite plant stimulants, to which the name
"hormones" has been applied. There is considerable experimental evidence
to support this conception that glucosides may be the source of stimulating
hormone substances, which will be discussed more in detail in the chapter
dealing with these plant stimulants (Chapter XVII).

Glucosides may also serve as the mechanism for putting out of action of
harmful products which may appear in the tissues as the result of abnormal
conditions. These harmful substances may be rendered soluble by

combination with sugars and so transposed by osmosis to some other part of
the plant. The abnormally large percentages of glucosides which are present
in certain species of plants during unfavorable climatic conditions lends
some support to this view.

Finally, it may be assumed that easily oxidizable substances, such as
aldehydes and acids, are possibly protected against too rapid, or premature,
oxidation by being transformed into glucosides.

In general, it may be said that the glucosides seem to serve as the
regulatory, protective, and sanatory agencies of the plant mechanism.

BIOLOGICAL SIGNIFICANCE OF GLUCOSIDES

The bitter taste of glucosides and their almost universal presence in the
bark of plants undoubtedly helps to prevent the destructive gnawing of the
bark by animals.

Glucosides having either a strong bitter taste, or pronouncedly poisonous
properties, likewise undoubtedly serve to protect such important organs of
plants as the seeds and fruits from being prematurely eaten by birds and
animals. The common disappearance of these bitter substances as the seed or
fruit ripens adds to the attractiveness of the material for food for animals at
the proper stage of ripeness to provide for wider distribution of the seeds for
further propagation. Further, the very general occurrence of these protective
glucosides in many of the vegetative parts of plants during the early stages
of growth, followed by their disappearance after the seeds of the plant have
been formed, certainly serves to protect these plants from consumption as
forage by animals before they have been able to develop their reproductive
bodies. The lack of palatability, and even the production of digestive

disorders resulting from the eating of unripe fruit may be due, in part at least,
to the presence of protective glucosides in unripe fruits and vegetables.

On the other hand, the almost universal presence of the brilliant pigment
glucosides in the external parts of flowers undoubtedly serves to attract the
insects which are biologically adapted to provide for the transportation of
pollen from one blossom to another and so to insure the cross-fertilization
which is so important in maintaining the vigor of many species of plants.

It is apparent that this important group of compounds, with its exceedingly
varied and complex constituent groups, may play a variety of significant
rôles in plant growth.

References.

Armstrong, E. F.—"The Simple Carbohydrates and Glucosides,"
239 pages, Monographs on Biochemistry, London, 1919 (3d ed.).

Van Rijn, J. J. L.—"Die Glykoside," 511 pages, Berlin, 1900.

CHAPTER VII

TANNINS

Using the term in its general application to a group of substances having
similar chemical and physical properties, rather than in its limited
application to a single definite chemical compound known commercially as
"tannin," the tannins are a special group of plant substances, mostly
glucosides, which have the following characteristic properties. First, they are
non-crystalline[4] substances, which form colloidal solutions with water,
which have an acid reaction and a sharp astringent taste. Second, they form
insoluble compounds with gelatine-containing tissues, as shown by the
conversion of hide into leather. Third, they form soluble, dark-blue or
greenish-black compounds with ferric salts, the common inks. Fourth, they
are precipitated from their solutions by many metallic salts, such as lead
acetate, stannous chloride, potassium bichromate, etc. Fifth, they precipitate
out of solution albumins, alkaloids, and basic organic coloring matters.
Finally, most tannins, in alkaline solutions, absorb oxygen from the air and
become dark brown or black in color.

FOOTNOTES:

[4 The needle-like forms, in which commercial "tannin" comes on the market, are not true
] crystals, but are broken fragments of the threads into which the colloidal tannin is "spun-
out" from the syrupy extracts of nutgalls, etc.

OCCURRENCE

Tannins occur widely distributed in plants. Practically every group of
plants, from the fungi up to the flowering plants, contains many species of
plants which show tannin in some of their tissues. Among the higher plants,
tannins occur in a great variety of organs. Thus, they are found in the roots
of several species of tropical plants; in the sterns, both bark and wood, of
oaks, pines, hemlock, etc.; in the leaves of sumac, rhododendron, etc.; in
many fruits, especially in the green, or immature, stages; and in the seeds of
several species, either before or after germination. Tannins are also found in
certain special structures, such as gland cells, cells of the pulvini, laticiferous
tissues, etc. Further, they are especially abundant in the pathological growths
known as galls, which often contain from 40 to 75 per cent of tannin and
constitute the most important commercial source for these materials.

The principal commercial sources of tannin, which is used in the
manufacture of inks, in the tanning of leather, in certain dyeing operations,
etc., are oak-galls, the bark and wood of oak, hemlock, acacia, and
eucalyptus, the bark of the mangrove, the roots of canaigre, and the leaves of
several species of sumac.

CHEMICAL CONSTITUTION

Tannins are either free phenol-acids or, more commonly, glucosides of
these acids. Common "tannin," when hydrolyzed, yields from 7 to 8 per cent
of glucose, which indicates that it is a penta-acid ester of glucose, i.e., each
glucose molecule has five acid groups attached to it. The formula for such a
tannin is, therefore, as follows,

in which the R represents a complex phenol-acid like tannic acid, or
digallic acid. These acids are derivatives of the common phenols, whose
constitution will be brought to mind by the following series of formulas:

These phenols themselves do not occur as constituents of tannins,
although they are often found in other glucosides, gums, etc. The following
mono-carboxyl acid derivatives of these phenols are, however, found both

free and in glucoside formation as constituents of many of the common
tannins.

Pyrocatechuic acid, derived from pyrocatechol, represented by the
formula,

Gallic acid, derived from pyrogallol, and represented by the formula,

In most of the common tannins, however, the characteristic acids are oxy-
derivatives of the so-called "tannon" group, represented by the formula,
C6H5·CO·O·C6H5. For example, digallic acid, which is a constituent of
many common tannins, is a tetra-oxy, mono-carboxyl derivative of this
group, having the structural formula,

Ellagic acid, which is an hydrolysis product of many of the pyrogallol
tannins (see below) and which produces the characteristic "bloom" on
leather tanned by this type of tannins, has the following formula,

CLASSES OF TANNINS

The tannins are divided into two general classes, known respectively as
the pyrogallol tannins and the catechol tannins. These differ in their
characteristic reactions as follows:

Pyrogallol variety Catechol variety
Ferric salts Dark blue Greenish black
Bromine water No precipitate Yellow or brown precipitate

Leather Produce a "bloom" No "bloom"
Conc. sulfuric acid Yellow or brown Red or pink
Lime water Gray or blue ppte. Pink to brown ppte.

Pyrogallol tannins contain approximately 52 per cent of carbon; while the
catechol tannins usually contain 59 per cent to 60 per cent, the difference
being due to the absence of glucose from the molecule in the latter types.

The two types are distributed in plants as follows: pyrogallol tannins in
oak-galls, oak wood, sumac, chestnut, divi-divi, and algaro billa; catechol
tannins in the barks of pines, hemlocks, oaks, acacias, mimosas, cassia, and
mangrove, in quebracho wood, canaigre roots, cutch and gambier. The so-
called "pseudo-tannins" (i.e., compounds which do not tan leather but
possess other properties like tannins) are found in hops, tea, wine, fruits, etc.

SOME COMMON TANNINS

Ordinary commercial "tannin," or "tannic acid," is a compound of one
molecule of glucose with five of digallic acid. It is found in many plants, and
is prepared commercially from the Turkish oak-galls and the Chinese sumac-
galls. It exhibits all the characteristic properties which have been listed
above for tannins in general and responds to all the characteristic reactions
of a pyrogallol tannin. It is extensively used for the manufacture of blue-
black ink, and in many technical processes.

Catechu tannin and catechin are compounds of the catechol tannin type.
The latter is obtained from acacia wood, mahogany wood, mimosa wood,
etc. It is not a true tannin, since it does not convert hide into leather; but
when heated to 120° or above, it is easily dehydrated, forming catechu
tannin which is identical with that which is obtained directly from gambier
and Bombay cutch (products made by evaporating water extracts from the

bark of various tropical trees). This latter is a true tannin, which is much
used in dyeing and other technical processes.

"Quercitannic acid," obtained from oak bark, etc., is likewise a catechol
tannin. It yields no glucose on hydrolysis.

A great many other tannins are known, and their possibilities for technical
use in tanning, dyeing, etc., have generally been investigated; but so little
has been learned about their composition and relation to the plant's own
needs, that it seems unnecessary to discuss them in detail here.

PHYSIOLOGICAL USES OF TANNINS

Tannins are probably not direct products of photosynthesis. They are,
however, elaborated in the green leaves of plants and translocated from there
to the stems, roots, etc. Their close association with the photosynthetic
carbohydrates has led many investigators to seek to establish for them some
significant function as food materials, or as plastic substances in cell
metabolism. Many conflicting views have been advanced, but a careful
review of these leads inevitably to the conclusion that tannins probably do
not serve in any significant way as food material. The glucose which is
generally present in the tannin molecule may, of course, serve as reserve
food material, but it seems probable that it functions as a constituent of the
tannins only to assist in making them more soluble and hence more easily
translocated through the plant tissues.

Some fungi, and perhaps other plants as well, can actually utilize tannins
as food material under suitable conditions and in the absence of a proper
supply of carbohydrates. But this does not prove that tannins can normally
replace carbohydrates as food material for these species of plants.

There seems to be ample evidence that tannins are elaborated where
intense metabolism is in progress, such as occurs in green leaves during the
early growing season; in the rapid tissue formation which takes place after
the stings of certain insects, producing galls, etc.; during germination, and as
a result of any other unusual stimulation of metabolism. It may be, therefore,
that tannins serve as safety accumulations of excessive condensations of
formaldehyde, or other photosynthetic products, under such conditions. It
seems certain that in all such cases tannins are the result of, and not (as some
investigators have supposed) the causative agents for, the abnormally rapid
metabolism.

It seems to be fairly well demonstrated that tannins are intermediate
products for the formation of cork tissue. This may account for their
common occurrence in the wood and bark of trees. Indeed, it has been
shown that gallic and tannic acids are present in considerable proportions in
those parts of the plant where cork is being formed. Further, that they bear
direct relation to cork-formation has been demonstrated in two different
ways. First, cork-like substances have been artificially produced by passing a
stream of carbon dioxide through mixtures of formaldehyde with various
tannic acids. Second, by various treatments of cork, decomposition
compounds showing tannin-like properties may be obtained.

Some investigators have held that not only cork tissue but also other
lignose, or cell-wall material, may be developed from tannins. Certain
observations with Spirogyra seem to indicate that tannin may play an
important part in the formation of new cell walls during conjugation, as cells
which are ready to conjugate are rich in tannin, which gradually diminishes
in quantity until it is practically absent at the time of spore-formation. There
seems to be no evidence that tannins perform any such function as this in
higher plants, however.

Again, tannins may play a very important part in pigment-formation. They
are very similar in structure to the anthocyanin pigments, both being made
up of practically identical decomposition units, the phenolic bodies. The
disappearance of tannins during the process of ripening of fruits may be
connected, in part at least, with the development of the brilliant red, blue,
and yellow pigments which give such rich colors to the thoroughly ripe
fruits.

Finally, certain of the tannins undoubtedly serve as protective agents to
prevent the growth of parasitic fungi in fruits, etc. Recent investigations
show that at least some of the varieties of fruits which are resistant to the
attacks of certain parasitic diseases utilize tannins for this purpose. This
protective effect may be accomplished in two different ways. Either the
tannin actually serves as an antiseptic to prevent the growth of the parasitic
fungus within the tissues of the host plant, or it assists in the development of
a corky layer which "walls-off" the infected area and so prevents further
spread of the disease through the tissue. Examples of both types of
protective action have recently been reported.

It is obvious that the different forms of tannins may play different rôles in
plant life, and the same tannin substance may possibly serve different
purposes under different conditions.

BIOLOGICAL SIGNIFICANCE OF TANNINS IN
FRUITS

The presence of tannins in fruits and the changes which they undergo
during the ripening process cannot fail to attract attention to their biological
significance in serving to protect the fruit from premature consumption as
food by animals.

Tannins are of frequent occurrence in green fruits, imparting to them their
characteristic astringent taste. They nearly always disappear as the fruit
ripens. The fact that during the ripening process both sugars and fruit esters,
as well as attractive surface pigments, are developed has led certain
investigators to the conclusion that tannins serve as mother-substances for
these materials in the green fruits and are converted into these attractive
agencies during ripening. There is nothing in the chemical composition of
tannins which indicates, however, that they are precursors of sugars or fruit
esters, although (as has been pointed out) they may give rise to anthocyan
pigments.

Further, recent researches concerning the tannin of persimmons (the best-
known and most striking example of the phenomena under discussion)
clearly show that the tannin is not actually used up during the ripening
process; that instead it remains in the ripe fruit in practically undiminished
quantity; but that when the fruit is ripe, the tannin is enclosed in certain
special large cells or sacs, which are surrounded by an insoluble membrane,
so that when the fruit is eaten by animals the astringent tannin, enveloped in
these insoluble sacs, passes by the organs of taste of the animal without
causing any disagreeable effects. This walling-off of the astringent tannins
can be stimulated in partially ripe fruits by treating them with several
different chemical agents, the simplest method being that of placing the
unripe fruit in an atmosphere of carbon dioxide gas for a short period. The
artificial "processing" of persimmons to render them edible for a longer
period before they become naturally fully ripe and subject to decay is now a
commercial enterprise. This process is of interest because of its possible
connection with the conversion of tannins into cork, under the influence of
carbon dioxide gas, as mentioned in a preceding paragraph.

From these facts, it is apparent that in persimmons, and probably in other
tannin-containing fruits, the process of natural selection has developed a

mechanism for the secretion of tannin in green fruits, followed by a process
for walling it off in harmless condition when the fruit is ripe, which serves
most admirably to protect the fruit from consumption by animals before the
enclosed seeds have fully developed their reproductive powers.

References.

Abderhalden, E.—"Biochemisches Handlexikon, Band 7,
Gerbstoffe, Flechtenstoffe, Saponine, Bitterstoffe, Terpene,
Aetherische Oele, Harze, Kautschuk," 822 pages, Berlin, 1912.

Allen's Commercial Organic Analysis, Vol. 5, "Tannins, Dyes
and Coloring Matters, Inks," 704 pages, 6 figs., Philadelphia,
1911 (4th ed.).

Cook, M. T. and Taubenhaus, J. J.—"The Toxicity of Tannin,"
Delaware College Agricultural Experiment Station Bulletin No.
91, 77 pages, 43 figs., Newark, Del., 1911.

Dekker, J.—"Die Gerbstoffe," 636 pages, 3 figs., Berlin, 1913.

Gore, H. C.—"Experiments on the Processing of Persimmons to
Render them Nonastringent," U. S. Department of Agriculture,
Bureau of Chemistry Bulletin No. 141, 31 pages, 3 plates, 1911;
and No. 155, 20 pages, 1912.

Lloyd, F. E.—"The Tannin-Colloid Complexes in the Fruit of the
Persimmon, Diospyros," in Biochemical Bulletin, Vol. 1, No. 1,
pages 7 to 41, 34 figs., New York, 1911.

CHAPTER VIII

PIGMENTS

Practically all plant structures contain pigments. These may be considered
as of two types: (a) the vegetative pigments, which have a definite energy-
absorbing rôle in the metabolic processes of the tissues which contain them,
and (b) the ornamental pigments. It is probable that the same chemical
compound may serve in either one of these capacities under different
conditions, but, in general, it is possible to assign either a definite vegetative,
or physiological, use, or else a simple ornamental, or biological, significance
to each of the common pigments. The first type is found widely distributed
through the protoplasm, or cell-sap, of the plant structures; while the
ornamental pigments are located chiefly in the epidermal cells, especially of
flowers.

With respect to their colors, the plant pigments may be grouped as
follows:

Green—the chlorophylls.
Yellow—the carotinoids, flavones, and xanthones.
Red—phycoerythrin, lycopersicin, anthocyanin.
Blue—anthocyan derivatives.
Brown—phycophæin, fucoxanthin.

Of these, the chlorophylls, the carotinoids, phycoerythrin (in red sea-
weeds) and phycophæin (in brown sea-weeds) are generally vegetative
pigments; while the others form the basis for most of the ornamental
pigments, although they may have a definite energy-absorbing effect, in
some cases.

THE CHLOROPHYLLS

The importance of the green coloring matter in plants has been understood
for more than a century, its connection with photosynthesis having been
known as far back as 1819. But definite knowledge as to its chemical
constitution is of very recent origin. As recently as 1908, it was asserted that
chlorophyll is a lecithin-like body, yielding choline and glycero-phosphoric
acid on hydrolysis. It is now known, however, that chlorophyll contains
neither choline nor phosphorus, the earlier observations being due to
mixtures of various other materials with the true chlorophyll in the extracts
which were examined. Beginning with 1912, Willstätter and his
collaborators, in a series of classic papers which were finally collected in
book form, clearly demonstrated the chemical constitution of the green
pigments of plants, which had been previously designated under the single
name "chlorophyll." In 1912, Willstätter and Isler first showed that the green
coloring matter which is extracted from plants by alcohol, ether, etc., is
made up of two definite chemical compounds, to which they assigned the
names "chlorophyll a" and "chlorophyll b," associated with two yellow
pigments, carotin and xanthophyll, and, in some cases, with the reddish-
brown fucoxanthin. The percentages of total pigment materials, and the
relative proportions of the five different pigments, in several types of plants,
are as follows:

Land Green
Brown
Plants, Algæ,
Seaweeds,
Per Per
Per Cent.
Cent. Cent.
Total pigment in the dry
0.99 0.29 0.21
matter
Proportion of:
Chlorophyll a 63 55 44
Chlorophyll b 22 4 31
Carotin 6 11 7
Xanthophyll 9 10 18
Fucoxanthin 20

The two chlorophylls have the following formulas: chlorophyll a,
C55H72O5N4Mg, and chlorophyll b, C55H70O6N4Mg. Hence, they differ only
in having two hydrogen atoms in the one replaced by one oxygen atom in the
other. Both are amorphous powders, from which crystalline chlorophyll (see
below) can be obtained by hydrolysis. Chlorophyll a is blue-black, is easily
soluble in most organic solvents, and when saponified by alcoholic potash
gives a transient pure yellow color. Chlorophyll b is dark green, is somewhat
less soluble than the other form, and when saponified by potash gives a
transient brilliant red.

Amorphous and Crystalline Chlorophyll.—When the chlorophyll of
plants is extracted by alcohol and the alcoholic extract evaporated nearly to
dryness, beautiful dark green crystals are obtained. Willstätter has shown,
however, that in these crystallized forms the ethyl group (from the ethyl
alcohol used) has replaced the phytyl group (see below) which is present in
the pigments as they exist in the plant tissues; and that, when extracted by
other solvents than alcohol, the pigments may be obtained in the amorphous
forms in which they exist in the plant.

This change from amorphous to crystalline compounds may be
understood from the preliminary statement that the chlorophylls are esters of
tri-basic acids, in which one acid hydrogen is replaced by the methyl (CH3)
group and a second by the phytyl (C20H39, from phytol, or phytyl alcohol,
C20H39OH) group. When treated with ethyl alcohol (C2H5OH) for the
purpose of extracting the pigments, the ethyl (C2H5) group replaces the
phytyl group, thus yielding a methyl-ethyl ester, and these esters are the
crystalline forms of the chlorophylls. This replacement is made possible
through the action on the original pigment in the tissues of an enzyme,
chlorophyllase, which is also present in the tissues, which splits off the
phytyl group, forming phytyl alcohol, and leaving a free COOH group in the
pigment, with which the alcohol used in the extraction forms the ethyl ester
(see Chapter IX for a discussion of the formation and hydrolysis of esters).

While the chlorophylls are tri-basic acids, only two of the acid COOH
groups actually function in ester-formation. The third acid group seems not
to exist as a free acid group; but in chlorophyll a, it is in what is known as
the "lactam" arrangement, represented by the —CONH— group, and in
chlorophyll b, it is probably in the "lactone" arrangement, represented by the
—COO— group; the two bonds in each case being attached to different
structural units in the molecule (see page 106).

The change from amorphous to crystalline forms may be represented by
the following formulas, in which the R represents the whole of the complex
group to which the acid ester groups are united:

"Chlorophyllin," the compound in which the ester groups have been
converted into free acid groups, as indicated above, may be obtained from
either amorphous or crystalline chlorophyll by treatment with caustic potash
dissolved in methyl alcohol.

Phytol.—This alcohol, which furnishes the characteristic ester group in
the chlorophyll of plants, is a compound of very unusual composition, which
has never been found in any other form or in any other type of compound
which is present in either plant or animal tissues. Careful studies of its
addition and oxidation products prove that it has the following structural
arrangement:

As this formula indicates, the compound contains one unsaturated,
double-bond linkage, one primary alcohol group, and eleven methyl groups.
As has been said, this alcohol occurs nowhere else in nature, and its presence
and function in the chlorophyll molecule are, as yet, wholly unexplainable.
Phytol itself is a colorless, oily liquid, with a high boiling point (145° in
vacuo, 204° at 10 mm. pressure).

THE CONSTITUTION OF THE CHLOROPHYLLS

As has been mentioned, chlorophyll a differs from chlorophyll b by
having one more oxygen and two less hydrogen atoms in the molecule, and
in having one of its nitrogen atoms in the "lactam" arrangement. These
differences in structure are represented by the following formulas which are

commonly used to represent the two compounds, but which do not show the
arrangements of the major groups of the complex molecules:

The chlorophylls are unstable compounds, readily acted upon by acids or
alkalies, and by the enzyme chlorophyllase, which splits off the phytyl
alcohol group. The progressive action of acids and of alkalies in breaking
down the molecule, and the products of its oxidation and reduction, have
served to establish the chemical composition of the compound in each case.
Because of the importance of these pigments in the whole metabolic
processes of the plant, it seems to be desirable to consider the nature of these
reactions in some detail, as follows:

Decomposition of the Chlorophylls by Alkalies.—The first action of
dilute alkalies on the chlorophylls is to split off, by hydrolysis, the alcoholic
groups of the esters, producing the crystalline tri-basic acids, or
chlorophyllins a and b. Each of these chlorophyllins exists in two forms, the
normal and the iso, in which the attachment of the COOH groups to the
other groups in the molecule is in different positions. Hence, chlorophyll a
yields chlorophyllin a and isochlorophyllin a, and chlorophyll b yields
chlorophyllin b and isochlorophyllin b, all four of which are tri-basic acids.

These compounds, when heated with alkalies, split off carbon dioxide in
successive stages, losing one COOH group at each step, thus yielding a
series of simpler compounds of the following types: First, di-basic acids;
second, monobasic acids; and finally, ætiophyllin, a compound in which no
COOH group is present. In all of these compounds, derived from
chlorophylls by the action of alkalies, the Mg remains in the molecule, and

all the Mg-containing derivatives from the chlorophylls are known as
"phyllins." At the stage at which only one COOH group remains in the
molecule, only one group arrangement is possible, and the derivatives from
chlorophyllin a and isochlorophyllin b, and those from chlorophyllin b and
isochlorophyllin a, are identical. At the final stage, the derivatives from all
four forms are identical. This may be graphically illustrated by the following
diagram indicating the progressive decomposition of the two chlorophylls
under the action of alkalies:

Decomposition of Chlorophylls by Acids.—The first action of dilute
acids upon chlorophylls is to remove the magnesium, without otherwise
changing the molecule. Two hydrogens go in in the place of the magnesium.
Dilute acids act in precisely the same way upon each of the "phyllins" shown
in the above scheme. In this way, a whole series of compounds,
corresponding to each of the chlorophylls and their alkali-decomposition
products, but with the magnesium lacking in each case, has been prepared.
Thus,

Similarly,

Isochlorophyllin a, becomes Phytochlorin e, Chlorophyllin a, becomes
Phytochlorin f, and g,

Isochlorophyllin b, becomes Phytorhodin g Chlorophyllin b, becomes
Phytorhodin i and k,

And bodies known as "porphyrins" are similarly derived from all the other
known phyllins.

For example: cyanophyllin, MgC31H32N4(COOH)2, becomes
cyanoporphyrin, C31H34N4(COOH)2; ætiophyllin, MgC31H34N4, becomes
ætioporphyrin, C31H36N4, etc.

Phytochlorin e and phytorhodin g are the chief products of the
decomposition by acids of the chlorophylls. Indeed, it was the production of
these compounds which led to the discovery of the existence of the two
chlorophylls. When treated with alkalies, they lose their carboxyl groups and
become ætioporphyrin.

Decomposition of the Chlorophylls by Oxidation and Reduction.—
When acted upon by oxidizing agents, such as chromic acid, the porphyrins
yield two chief oxidation products, which are pyrrole derivatives having the
following formulas,

By reduction, there have been obtained from the chlorophylls and the
various porphyrins, three isomeric pyrrole derivatives having the following
formulas,

As a result of the study of these decomposition units, Willstätter has
suggested the following formulas for the structural arrangement of
ætiophyllin and ætioporphyrin, the compounds which result from the
removal of all of the acid groups and finally of the magnesium from the
chlorophylls,

The COOH groups which are attached to these compounds to form the
various phyllins and porphyrins, as well as the original chlorophylls, are
supposed to be attached to the C2H5 groups in the above formulas, the
different modifications, or compounds, depending upon the position in
which one or more of these attachments are made.

SIMILARITY OF CHLOROPHYLL AND
HÆMOGLOBIN

It seems to be desirable, at this point, to call attention to the remarkable
similarity in the chemical composition of chlorophyll, the most important
pigment of plants, and hæmoglobin, the all-important respiration-regulating
pigment in the blood of animals. Hæmoglobin is a complex compound,

consisting of about 96 per cent of albumin (a protein, see Chapter XIII)
united with about 4 per cent of hæmatin, a brilliant red pigment which has
the formula FeClC32H32O4N4. When treated with acids, the iron (and its
accompanying Cl) is removed, and hæmatoporphyrin, C32H36O4N4, is
obtained. When either hæmatin, or hæmatoporphyrin is oxidized, hæmatinic
acid imide identical with that obtained from ætioporphyrin is obtained. Also,
when hæmatoporphyrin is reduced, hæmopyrrole identical with that from
ætioporphyrin is obtained. Thus, it would appear that the unit structural
groups in hæmatin and in chlorophyll are identical; although chlorophyll
may exhibit more variations in isomeric arrangement of these structural units
than have been found in hæmatin. Hence, it is apparent that the only
essential difference in composition between chlorophyll and hæmatin is that
in the former the structural units are linked together by iron, while in the
latter, the same units are united through magnesium as the linking element.
Further, it is known that while iron is not a constituent element in the
chlorophyll molecule, it is, in some unknown way, absolutely essential to the
production of chlorophyll in plants; plants furnished with an iron-free
nutrient solution rapidly become etiolated and photosynthesis stops.

The following skeleton formulas have been suggested to indicate the way
in which these elements are linked between the structural units in their
respective compounds.

It is understood, of course, that the mineral element does not furnish the
definite means of holding the structural units together as otherwise it would
not be possible to remove the iron, or magnesium, without breaking down
the molecule, as is done in the case of the porphyrins. The actual binding
linkage is undoubtedly between carbon atoms, as indicated in Willstätter's
formulas for ætiophyllin and ætioporphyrin (see page 109). The attachment
of the magnesium to each one of the four nitrogen atoms in the skeleton
formula assumes the existence of subsidiary valences of 2-4 for magnesium
(and of 3-5 for iron), or of possible oscillating valences similar to those
supposed to be exhibited by carbon in its closed-ring arrangements.

PROPERTIES OF THE CHLOROPHYLLS

The phytyl esters, or natural chlorophylls, are amorphous solids; while the
methylethyl esters (chlorophyllins) and the free acids (phyllins) are
crystalline compounds. All of these compounds are easily soluble in ether
and alcohol, but insoluble in water. The chlorophylls and chlorophyllins are
practically insoluble in petroleum ether and chloroform; but the monobasic
acids (pyrrophyllin and phyllophyllin) and the neutral ætiophyllin dissolve
easily in chloroform.

Solutions of the chlorophylls are fluorescent, being green by transmitted,
and red by reflected light.

Chlorophyll a is a blue-black solid, which gives dark green solutions in all
of its solvents. Chlorophyll b is a dark-green solid, which yields brilliant
green solutions. Solutions in ether of glaucophyllin and of cyanophyllin are
blue; of rhodophyllin, deep violet; of rubiphyllin, light violet; of
erythrophyllin, red; and of pyrrophyllin and phyllophyllin, bluish-red.

Solutions of the porphyrins are all red, the di-basic ones being usually a
bluish-red, and the simpler ones a brilliant red to deep brownish-red in color.

The several chlorophyll derivatives are further distinguished by
characteristic differences in their absorption spectra. These differences have
been pictured by Willstätter in his book dealing with the results of his
investigations concerning the chlorophylls, and reproduced in one or two
other texts which treat in detail with the physical-chemical properties of
these pigments, but need not be presented in such detail here.

THE CAROTINOIDS

The characteristic brilliant green of healthy plant tissues is due to the fact
that there are always associated with the dark bluish-green chlorophylls two
(or more) yellow pigments. These are known as the "carotinoids." This
group includes the two brilliant yellow pigments, carotin and xanthophyll,
and the reddish brown fucoxanthin and the brilliant red lycopersicin, which
are similar in their chemical composition. The first two are found universally
distributed in plants, associated with the chlorophylls, and may be regarded
as vegetative pigments, although the characteristic ornamental yellow and
orange colors of many flowers and fruits, as well as that of the roots of
carrots, etc., due to these pigments.

Carotin.—This pigment occurs in various forms in plants, both
amorphous and crystalline. It crystallizes out of solution in flat plates, which
are orange-red by transmitted light, and greenish-blue by reflected light, and
have a melting point of 168°. Carotin is insoluble in water, only very slightly
soluble in acetone or cold alcohol, readily soluble in petroleum ether, ether,
chloroform, and carbon disulfide. Its solutions are strongly fluorescent.

Its molecular formula is C40H56. It is, therefore, a hydrocarbon of a very
high degree of unsaturation. On exposure to dry air, it absorbs 34.3 per cent
of its own weight of oxygen, which corresponds to 11-1/2 atoms of oxygen,
computed on the basis of the molecular formula C40H56, and would indicate
a formula of (C40H56)2O23 for the oxygenated compound; this being three
oxygen atoms less than would be required to bring the compound to the
theoretical stage of saturation represented by the unimolecular formula
CnH2n+2. In moist air, two more oxygen atoms are absorbed, probably
forming two OH groups in the molecule. Moreover, carotin absorbs iodine.
When the calculated amount of iodine is used, a definite compound having
the formula C40H56I2 is produced; but in the presence of an excess of iodine
another compound having the apparent formula C40H56I3 (or 2C40H56I2+I2)
is obtained. (Note that 2 atoms of iodine plus 12 atoms of oxygen, or 3 of
iodine plus 11-1/2 of oxygen, produce the degree of saturation required by
the formula CnH2n+2.) It is evident from these experimental data, that a part
of the unsaturated linkage in the carotin molecule is of a type which can
easily be saturated by direct addition of oxygen, while the remainder may be
saturated by iodine.

The reaction of carotin toward bromine is peculiar. With this element, it
forms a compound having the formula C40H36Br22, indicating the direct
addition of two atoms of bromine and the substitution of twenty atoms of
this element for the same number of hydrogen atoms.

The oxygenated carotins are colorless substances, while the iodide
crystallizes in beautiful dark-violet prisms, having a coppery red
fluorescence.

Xanthophyll is closely related to carotin. It has the molecular formula
C40H56O2. It absorbs 36.55 per cent of oxygen (corresponding to 13 atoms,
which would indicate the formation of two OH groups in addition to the

saturation required by the CnH2n+2 formula); and an iodine addition product
having the formula C40H56O2I2, which crystallizes in dark-violet needles.

Xanthophyll differs markedly from carotin in its solubilities, being
insoluble in petroleum ether and only sparingly soluble in carbon disulfide.
It may be fairly easily reduced to carotin. This transformation is reversible,
and suggests a similarity to the change from hæmoglobin to
oxyhæmoglobin, and the reverse, in the blood of animals, as a part of their
respiration process.

Separation of the Chlorophylls, Carotin, and Xanthophyll.—These
pigments, which exist together in most plant tissues, may easily be separated
from each other by taking advantage of the differences in their solubilities,
according to the following procedure. Grind up a small quantity of the fresh
tissue (leaves of the stinging nettle furnish a conveniently large supply of
each of these pigments) with fine sand in a mortar. Cover with acetone, let
stand a few moments and then filter on a Büchner funnel. Pour the filtrate
into a separatory funnel, add an equal volume of ether and two volumes of
water. Shake up once and then allow the ether layer to separate; the pigments
will be in this layer. Drain off the water-acetone layer. Now to the etherial
solution, add about half its volume of a concentrated solution of potassium
hydroxide in methyl alcohol. Shake well and allow to stand until the mixture
becomes permanently green. Now add an equal volume of water and a little
more ether, until the mixture separates sharply into two layers. The
chlorophylls will now be in the lower dilute alcohol layer, and the
carotinoids in the upper ether, and may be separated by draining of each
layer separately. To separate the carotin from xanthophyll place the ether
solution in a small open dish and evaporate to a small volume. Now add
about ten volumes of petroleum spirit and an equal volume of methyl
alcohol, stir up well, transfer to a separatory funnel and allow the two layers
to separate. The carotin will now be in the upper layer of petroleum ether,

and the xanthophyll in the lower alcohol layer; these layers may be drained
off separately and the solvents evaporated in order to recover the pigments in
dry form.

Lycopersicin (or lycopin) is a hydrocarbon pigment having the same
formula as carotin. It is, however, brilliantly red in color, and crystallizes in a
different form and has a different adsorption spectrum from carotin. It is the
characteristic pigment of red tomatoes, and is found also in red peppers.
Yellow tomatoes have only carotin as their skin-pigment, while lycopersicin
is usually present in the flesh of the ripe fruits of all varieties and in the skin
of red ones. It has been shown, however, that if varieties of tomatoes which
are normally red when ripe, are ripened at high temperatures, 90° F. or
above, their skins will be yellow instead of red when fully ripe. Hence, the
occurrence of carotin, or of lycopersicin, as the skin pigment is determined
in part by the varietal character (being different in different varieties when
ripened at normal temperatures) and in part by the temperature at which the
fruit ripens. The two pigments are, of course, isomers; but the difference in
their structural arrangement is not known.

Fucoxanthin, C40H54O6, is a brownish-red pigment, found in fresh brown
algæ, and in some brown sea-weeds. Its formula indicates that it is an
oxidized carotin. With iodine, it forms a compound having the formula
C40H54O6I4. It is unlike carotin and xanthophyll in that it has basic
properties, forming salts with acids, which are blue in color.

PHYCOERYTHRIN AND PHYCOPHÆIN

These are the principal pigments of red and brown seaweeds, respectively.
Their most characteristic difference from the pigments of non-aquatic plants
is that they are easily soluble in water, and insoluble in most organic

solvents, such as alcohol, ether, etc. At first thought, this would appear to be
impossible, since the plants grow in water and it would seem that their
water-soluble pigments would be continuously dissolved out of the tissues.
The reason why this does not occur lies in the fact that these pigments exist
in the cells of the seaweeds in colloidal form (see Chapter XV), and, hence,
cannot diffuse out through the cell-wails. The only way in which they can be
extracted from the tissues is by rupturing the cells, by grinding with sharp
sand, etc., after which the pigments can readily be dissolved out by water.

Phycoerythrin is the red pigment. It is a colloidal, nitrogenous substance,
allied to the proteins (see Chapter XIII) but not a true protein compound.
Hydrolysis by acids indicates that it contains leucine and tyrosine, two
amino-acids which are constituents of proteins, along with other bodies of
unknown composition.

The colloidal solution of phycoerythrin in water has a brilliant rose-red
color, with an orange fluorescence. It readily sets to a gel (see Chapter XV),
so that the solution is almost impossible to filter. On this account, purified
solutions of this pigment are very difficult to secure, and no satisfactory
analysis to indicate its composition has yet been obtained.

Actinically, it is a complementary pigment to chlorophyll, that is, it
absorbs the blue and green rays and permits the passage of light which is of
the wave length that is absorbed by chlorophyll.

Phycophæin.—Still less is known of the composition of this pigment than
of that of phycoerythrin. It is the characteristic pigment of brown seaweeds.
It is supposed to exist in the cells of algæ, chiefly as a colorless chromogen,
which becomes first yellow and then brown on exposure to air. Associated
with it are other pigments, which have been variously reported as carotin,
phycoxanthin, etc.

THE ANTHOCYANS

These are a group of pigments of red, blue, or violet color, which occur in
the flowers, fruits, or leaves of many species of plants. They are essentially
ornamental pigments, and constitute a large proportion of the brilliant colors
of flowers, etc. They occur not only dissolved in the cell-sap, but also as
deposits of definite crystals or amorphous compounds in the cell protoplasm.

They are all glucosides. When the anthocyans are hydrolyzed, the sugar
molecules are split off and the characteristic hydroxy-derivatives of the
three-ring anthocyan nucleus (figured on page 83), known as
"anthocyanidins," remain. These anthocyanidins are themselves pigments.
They have been shown to be all derivatives of the anthocyan nucleus. The
oxygen atom in this nucleus is very strongly basic and exhibits its
quadrivalent property by forming stable salts by direct addition of acid
radicles. The variation of color of the anthocyanins has been explained by
Willstätter, as follows; the red is the acid salt, the blue is a neutral metallic
salt, and the violet is the anhydride of the anthocyanidin in question, thus

All of the natural anthocyanin pigments appear to contain a chlorine atom
attached directly to the ring oxygen, as shown in the above partial formulas.
In addition, they have four, five, or six hydroxyl (OH), or methoxy (OCH3),
groups attached at various points around the three rings. The following
formula for œnidin, one of the most complex of these anthocyanidins, will
illustrate their structural arrangement.

Delphinidin is the corresponding compound without the two CH3 groups;
while cyanidin contains only five OH groups; and pelargonidin, only four
OH groups.

The anthocyanin pigments are soluble in water, alcohol, and ether, the
solutions being red or blue in color according to the acidity or alkalinity of
the medium. Their presence in many species of plants is hereditable, as these
plants come true to color from seed, as in the case of red beets, red cabbage,
several species of blue berries, etc. In other cases, the anthocyanin
development depends largely upon the conditions of growth, particularly
those which prevail during the later stages of development: as in the case of
apples, where the amount of red color in the skin depends to a large extent
upon the conditions under which the fruit ripens.

Anthocyanin pigments often make their appearance late in the season; in
fruits, etc., as the result of the normal ripening process but in leaves as the
result of shorter daylight illumination accentuated also by sharp frosts.

THE ANTHOXANTHINS

The yellow plant pigments, other than the carotinoids, are almost without
exception glucosides having a xanthone or flavone nucleus. These typical
nuclei are illustrated on page 83. In these nuclei, as in the anthocyan one, the
oxygen atom is strongly basic and combines with mineral acids to form salts
(the oxygen becoming quadrivalent) and the color of the pigment depending
upon the nature of the combination formed in this way.

The anthoxanthin pigments are yellow, crystalline solids, which are only
slightly soluble in water. They dissolve readily in dilute acids and alkalies,
giving yellow or red solutions which are of the same color in either acid or
alkaline media. They are extensively used as yellow dyes.

Many of the common members of this group have been mentioned in the
chapter dealing with the glucosides. The characteristic pigment nucleus of
several of these is as follows:

Chrysin, found in various species of poplar and mallows,

Apigenin, found in parsley and celery, as the glucoside apiin,

Campferol, found in Java indigo, as the glucoside campferitrin,

Fisetin, found in quebracho wood and fiset wood,

Quercitrin, found in oak bark, horse-chestnut flowers, and in the skin of
onions,

Morin, found in yellow wood (Morus tinctoria).

Gentisin, found in yellow gentian (Gentiana lutea),

As a rule, the most brilliant of these yellow pigments are found in the
largest quantities in the bark and wood of various species of tropical plants;
although they are also present, in smaller amounts, in the blossoms of
species growing in temperate zones.

The anthoxanthins are easily converted into anthocyanins, and vice versa,
by the action of oxidizing and reducing enzymes which are commonly
present in the tissues of the plants which develop the pigments.

THE PRODUCTION OF ORNAMENTAL
PIGMENTS IN FLOWERS, ETC.

The breeding of flowering plants having blossoms of almost any desired
color has become a commercial enterprise of large importance. The results
which have been obtained, in many cases, have been made the object of
scientific study of the genetics of color inheritance. These studies have
developed certain interesting facts with reference to the chemistry of the
development of these ornamental pigments, which may be briefly mentioned
here.

In many of the plants which have been studied, the color of the flowers
depends upon several different factors, as follows:

C, a chromogen (or color-producing substance) which is generally a
flavone or xanthone glucoside, and which may be either yellow or colorless.

E, an enzyme which acts upon C, to produce a red pigment.

e, another enzyme which acts upon the red pigment, changing it to some
other anthocyanin color.

A, an antioxidase, or antienzyme, which prevents the action of E.

R, an enzyme which changes reds to yellows.

Thus, if a plant whose flower contains only the factor C be crossed with
one which contains the factor E, a red blossom will result, or if it contains
the factor e more intense pigments are developed. But if either A or R are
present, no change in the color of the original parents will result from the
crossing.

THE PHYSIOLOGICAL USES OF PIGMENTS

The vegetative pigments undoubtedly serve as agencies for regulating the
rate of metabolic processes. At the same time, it is extremely difficult to
determine whether the presence of a pigment in any given case is the cause
or the effect of the changes in the plant's activities which result from changes
in its external environment.

The chlorophylls are, of course, the regulator of photosynthesis, absorbing
solar energy with which the photosynthetic process may be brought about.
The simultaneous presence of carotinoids in varying amounts undoubtedly
serves to modify the amount and character of the radiant energy absorbed, as
these pigments absorb a different part of the spectrum of light and hence
undoubtedly produce a different chemical activity or "actinic effect" of the
absorbed energy. The variations in depth of color of foliage during different
growing conditions, from a pale yellow when conditions are unfavorable and
growth is slow to the rich dark green of more favorable conditions, is a
familiar phenomenon. Whether this change in pigmentation is the result of
an adjustment of the plant protoplasm, so that it can absorb a more highly
actinic portion of the light, or is a direct effect of the lack of conditions
favorable to chlorophyll-production and active photosynthesis, has not yet
been determined.

But there must be some influence other than response to environmental
conditions which controls the vegetative color in plants, since shrubs, or
trees, which have green, yellow, red, and purple leaves, respectively, will
grow normally, side by side, under identical external conditions of sunlight,
moisture supply, etc. The hereditary influence must completely overshadow
the apparent normal self-adjustment of pigment to energy-absorbing needs,
in all such cases.

Again, it appears that there is some definite connection between pigment
content and respiration. It is known, of course, that the gaseous exchanges
involved in animal respiration are accomplished through the reversible
change of hæmoglobin to oxyhæmoglobin, these being the characteristic
blood pigments. The easy change of carotin, C40H56, to xanthophyll,
C40H56O2, and vice versa, and the reversible changes of the yellow
anthoxanthins to the red anthocyanins, under the influence of the oxidizing
and reducing enzymes which are universally present in plants, would
indicate the possibility of the service of these pigments as carriers of oxygen
for respiratory activities in plants in a way similar to that in which the blood
pigments serve this purpose in the animal body. The fact, which has been
observed in connection with the experimental studies of the development of
the lycopersicin, that tomatoes which normally would become red remain
yellow in the absence of oxygen, indicates that this pigmentation, at least, is
definitely connected with oxygen supply; and the further fact that the
development of lycopersicin in red tomatoes, red peppers, etc., is dependent
upon the temperature at which the fruit ripens, may indicate a definite
connection of this pigment with the need for more oxygen (or for more heat,
as suggested in the following paragraph) at these lower temperatures.

Again, many investigators have concluded that at least one function of the
anthocyanin pigments is to absorb heat rays and so to increase transpiration
and other chemical changes. In support of this view, there may be cited the

general presence of such pigments in arctic plants, their appearance in the
leaves of many deciduous trees after a frost in the fall, etc. Indeed, there is
much to support the view that the autumnal changes in foliage pigments
have the physiological function of absorbing heat in order to hasten the
metabolic processes of ripening and preparation for winter defoliation. The
rapid and brilliant changes in foliage coloring after a sharp frost which kills
the tissues and makes rapid translocation of the food material of the leaves to
the storage organs immediately necessary, have been explained as the
response of the pigmentation of the leaves to the need for increased heat-
absorption. On the other hand, the red pigments of the beet-root, etc., which
seem to be identical in composition with the other anthocyanin pigments,
can have no such function as those which have just been described.
Furthermore, the fact that the pigment often varies in color from red to
yellow or brown, depending upon the temperature under which the tissue is
ripening, makes it an open question whether the pigment is the regulating
agency or whether its nature is the result of the environmental conditions.
Or, in other words, it is a question whether these changes in color are a
mechanism by which the plant cell adjusts its absorptive powers, or whether
they are only the inevitable result of the changes in temperature upon a
pigment material which is present in the cell for an entirely different use.

A very interesting side-light upon the color changes which many species
of plants undergo when the external temperature falls has been shown by the
investigations of the relation of the sugar content of the plant tissues to their
pigmentation. It is a well-known fact that not only do many species of
deciduous plants show the characteristic reddening of their leaves after frost
in the autumn but also many evergreens (Ligustrum, Hedera, Mahonia, etc.)
exhibit a marked reddening, or purpling, of their foliage during the winter
months, with a return to the normal green color in the spring. Earlier
investigations, which have been confirmed by several repetitions, showed
that the red or purple leaves always contain higher percentages of sugar than

do green ones of similar types. More recent studies have shown that artificial
feeding of some species of plants with abnormally large portions of soluble
sugars produces a reddening of the foliage tissues which is apparently
identical with that which these tissues undergo as the result of low
temperatures. Thus, the connection between the natural winter reddening of
foliage and the development of sugar in the tissues during periods of low
temperatures (see page 64) seems to be clearly demonstrated. It appears that
at least a part of the seasonal changes in color of plants is either the cause of,
or the effect of, variations in sugar content of the tissues of the plants,
accompanying the changes in external temperatures.

Oftentimes, the anthocyanin pigments seem to be associated with sugar
production, as contrasted with the chlorophylls, which seem to be more
favorable to the production of starch. But in this case also, it is impossible to
say whether the pigment is the direct causative agent in the type of
carbohydrate production or whether it is the effect of the same external
factors which determine, or modify, the character of the carbohydrate
condensation.

BIOLOGICAL SIGNIFICANCE OF
ORNAMENTAL PIGMENTS

The ornamental pigments undoubtedly have definite biological
significance. When present in the storage roots, such as beet-roots, carrots,
etc., or in the above-ground parts of plants, they may have served to protect
these organs against herbivorous animals which were accustomed to
consume green foods.

In flowers, the brilliant ornamental pigments undoubtedly serve to attract
the insects which visit these blossoms in search of nectar, and in so doing

promote cross-fertilization. Recent experiments have demonstrated that
colors are much more efficient than odors in attracting insects.

Taken altogether, it is apparent that the pigments may have a variety of
important rôles in plants. At the same time, some of them may be waste
products, with no definite use in the plant economy.

References.

Abderhalden, E.—"Biochemisches Handlexikon, Band 6,
Farbstoffe der Pflanzen- und der Tierwelt," 390 pages, Berlin,
1911.

Perkin, A. G. and Everest, A. E.—"The Natural Organic
Colouring Matters," 655 pages, London, 1918.

Wakemen, Nellie A.—"Pigments of Flowering Plants," in
Transactions of the Wisconsin Academy of Sciences, Arts, and
Letters, Vol. XIX, Part II, pages 767-906, Madison, Wisc., 1919.

Watson, E. R.—"Colour in Relation to Chemical Constitution,"
197 pages, 65 figs., 4 plates, London, 1918.

Wheldale, M.—"The Anthocyan Pigments of Plants," 304
pages, Cambridge, 1916.

Willstätter, R. and Stoll, A.—"Untersuchung über
Chlorophyllen, Methoden und Ergebnisse," 432 pages, 16 figs.,
Berlin, 1913.

CHAPTER IX

ORGANIC ACIDS, ACID SALTS, AND ESTERS

Organic acids, either in free form, or partially neutralized with calcium,
potassium, or sodium, forming acid salts, or combined with various alcohols
in the form of esters, are widely distributed in plants. They occur in largest
proportions in the fleshy tissues of fruits and vegetables, where they are
largely responsible for the flavors which make these products attractive as
food for men and animals. But organic acids and their salts are also found in
the sap of all plants, and undoubtedly play an important and definite part in
the vital processes of metabolism and growth.

CHEMICAL CONSTITUTION

All organic acids contain one (or more) of the characteristic acid group,

—COOH, or , known as "carboxyl." This group is monovalent,
and in the simplest organic acid, formic acid (H2CO2), it is attached to a
single hydrogen atom, thus, H·COOH. In all other monobasic acids, it is
attached to some other monovalent group, usually an alkyl radical, i.e., a

radical derived from an alcohol and containing only carbon and hydrogen (as
methyl, CH3, ethyl, C2H5, butyl, C4H9, acryl, C2H3, etc.). Hence, the general
formula for all monobasic organic acids is R·COOH, the R representing any
monovalent radical. In the simplest dibasic acid, oxalic (H2C2O4), two
carboxyl groups are united to each other, thus, HOOC·COOH; but in the
higher members of the series, the two characteristic acid groups are united
through one or more —CH2— groups, or their oxy-derivatives (as
HOOC·CH2·COOH, malonic acid; HOOC·CH2·CH2·CH2·COOH, glutaric
acid; HOOC·CHOH·CH2·COOH, malic acid, etc.). Polybasic acids,
containing three or more carboxyl groups, linked together through one or
more alkyl carbon atoms, are also possible, and a few typical ones (as

are found in fruits and other plant tissues.

The H atom of the COOH group may be replaced by metals, in exactly the
same way as it is replaceable in inorganic acids, producing either neutral or
acid salts, depending upon whether all or only a part of the acid H atoms are
replaced by the basic element.

Similarly, the acid H atom of either an organic or an inorganic acid may
be replaced by the alkyl group of an alcohol, producing "ethereal salts," or
"esters."

Thus, with nitric acid;

NO2OH (HNO3) + C2H5OH = NO2OC2H5 (C2H5NO3) + H2O
Nitric acid Ethyl alcohol Ethyl nitrate

And, with acetic acid;

CH3·COOH (H4C2O2) + C2H5OH = CH3·COOC2H5 + H2O
Acetic acid Ethyl acetate

With dibasic or polybasic acids, either one or more of the carboxyl H
atoms may be replaced with an alcohol radical, so that both acid and neutral

esters of all such acids are possible. Examples of all of these different types
of derivatives of organic acids are frequently found in plant tissues.

The occurrence, properties, and functions of a particular type of glycerol,
and other esters of organic acids, which are known as fats and waxes, are not
taken into consideration in the following discussions, but reserved for a
subsequent chapter dealing specially with them.

SOME COMMON ORGANIC ACIDS

Free organic acids, or their mineral salts or volatile esters, sometimes
occur as separate and characteristic individual compounds in particular
species of plants, or fruits; but much more commonly, two, three, or even
more acids or their derivatives, are associated together.

Formic acid, H·COOH (H2CO2), occurs in free form and in considerable
proportions in the leaves of several species of nettle, where it is responsible
for the unpleasant effects of the "sting." It may be detected in small amounts
in the vegetative parts of many, if not all, plants, especially during periods of
rapid growth, and is probably one of the intermediate products in the
photosynthesis of carbohydrates (see Chapter III).

Higher members of the formic acid series (as acetic, CH3·COOH;
propionic, C2H5·COOH; butyric, C3H7·COOH; etc.) are often found in
small quantities in the leaves of many plants and seem to be
characteristically present in certain species. They are easily produced from
carbohydrates by bacterial action and, hence, are always present in
fermenting tissues, such as silage, sauerkraut, etc. Furthermore, the glycerol
esters of higher members of this and other monobasic acid series are
constituents of all natural fats and oils (see Chapter X).

Oxalic acid, HOOC·COOH (H2C2O4), is found in small amounts in
nearly all plants and in relatively large proportions in those of Oxalis,
rhubarb, etc. It occurs both as the free acid and as neutral, or acid, oxalates
of calcium, potassium, and, perhaps, of magnesium and sodium. Solid
crystals of insoluble calcium oxalate are often found in plant cells, and it has
been shown that when so deposited the calcium cannot become again
available for metabolic uses. It is stated, further, that such crystals form only
when calcium is in excess in the plant sap; hence, the deposition of
crystallized calcium oxalate seems to be a device for the avoidance of
excessive calcium rather than excessive oxalic acid, in the plant juices.

Succinic acid, HOOC·CH2·CH2·COOH (H6C4O4), occurs in many fruits
and vegetables, and is also found in some animal tissues. In fruits, it is
usually associated with its derivatives, malic and tartaric acids.

Malic acid, HOOC·CH2·CHOH·COOH (H6C4O5), occurs in apples and
in many small fruits, and in many vegetables. Acid calcium malate is now
produced commercially as a by-product from the manufacture of syrups
from fruit juices, and is used as a substitute for "cream of tartar" in the
manufacture of baking powders.

Tartaric acid, HOOC·CHOH·CHOH·COOH (H6C4O6), is found in many
fruits, but most characteristically in the grape, where it occurs as the mono-
potassium salt. During the fermentation of grape juice into wine, this salt is
deposited in considerable quantities in the bottom of the wine-casks. This
crude product is collected and sold under the name "argols." From these
argols, pure acid potassium tartrate is obtained by decolorization and
recrystallization, and constitutes the "cream of tartar" of commerce.

Citric Acid, , occurs in large
proportions in lemons, and associated with malic acid in strawberries,
cherries, currants, etc. It is also found in small quantities in the seeds of the
common leguminous vegetables, beans, peas, etc.

Tannic acid occurs widely distributed in the plant kingdom as a
constituent of the special type of glucosides known as tannins, whose
properties and functions have already been discussed (see Chapter VII).

PHYSIOLOGICAL USES OF ORGANIC ACIDS

No conclusive evidence concerning the rôle of organic acids in plant, or
animal, growth, has yet been produced. There can be no doubt that the
hypothetical carbonic acid and its acid and normal salts have a significant
effect in regulating the acidity or alkalinity of plant juices, or body fluids,
and so determining the nature of the enzymic activities and colloidal
conditions of the biological systems (see Chapters XIV and XV). It is
probable that other organic acids, such as formic, acetic, oxalic, and succinic
acids, in plants and sarco-lactic acid, in animal tissues, perform similar
regulatory rôles; but there seems as yet to be no indication as to why
different acids should be used for this purpose by different species, or
organisms; or as to the methods by which they perform their specific
functions, whatever these may be.

In plants, the organic acids are usually in solution in the sap. When the
plant ripens, they generally disappear, either being neutralized by calcium, or
other bases, and deposited as crystals in the leaves or stems, or else used up
in the synthesis of other organic compounds. Small proportions of these
acids are usually present in mature seeds, and the percentage increases

materially during germination, indicating that they play an important rôle in
insuring the proper conditions for the conversion of the reserve food of the
seed into soluble materials available for the nutrition of the young growing
plant.

BIOLOGICAL SIGNIFICANCE OF FRUIT ACIDS,
ETC.

The occurrence of organic acids, or their derivatives, which have
pronounced odors or flavors, in the flesh surrounding the seeds of fruits, in
the endosperm of vegetable seeds, or in the tubers, etc., of perennial plants,
thus making them attractive as food for animals and men, undoubtedly
serves to insure a wider distribution of the reproductive organs of these
plants; a fact which has unquestionably had a marked influence upon the
survival of species in the competitive struggle for existence during past eras
and in the development and cultivation of different species by man. Indirect
evidence that the proportion of these attractive compounds present in certain
species may have been considerably increased by the processes of "natural
selection" in the past is furnished by the many successful attempts to
increase the percentage of such desirable constituents in fruits or vegetables
by means of artificial selection of parent stocks by skillful plant breeders.

CHAPTER X

FATS AND OILS, WAXES, AND LIPOIDS

Included in this group are several different kinds of compounds which
have similar physical properties, and which, in general, belong to the type of
organic compounds known as esters, i.e., alcoholic salts of organic acids.
The terms "oil," "fat," and "wax," are generally applied more or less
indiscriminately to any substance which has a greasy feeling to the touch and
which does not mix with, but floats on, water. There are many oils which are
of mineral origin which are entirely different in composition from natural
fats. These have no relation to plant life and will not be considered here.

The natural fats, vegetable oils, and plant waxes are all esters. There is no
essential difference between a fat and an oil, the latter term being usually
applied to a fat which is liquid at ordinary temperatures. The waxes,
however, are different in chemical composition from the fats and oils, being
esters of monohydric alcohols of high molecular weight, such as cetyl
alcohol, C16H33OH, myristic alcohol, C30H61OH, and cholesterol,
C27H45OH; whereas the fats and oils are all esters of the trihydric alcohol
glycerol, C3H5(OH)3. Lipoids are much more complex esters, having some
nitrogenous, or phosphorus-containing, group and sometimes a sugar in

combination with the fatty acids and glycerol which make up the
characteristic part of their structure.

In general, waxes and lipoids have a harder consistency than fats: but this
is not always the case, since "wool-fat" and spermaceti, both of which are
true waxes in composition, are so nearly liquid in form as to be commonly
called fats; while certain true fats, like "Japan wax," are so hard as to be
commonly designated as waxes. It is plain that physical properties alone
cannot be relied upon in the classification of these bodies. In fact, there is no
single definite property by which members of this group can be accurately
identified. There are many other types of substances belonging to entirely
different chemical groups, which have oily, or fat-like, properties.

A. FATS AND OILS

OCCURRENCE

Fats and oils are widely distributed in plants. They occur very commonly
in the reproductive organs, both spores and seeds, as reserve food material.
In fungi, oils are often found in the spores, but sometimes also in sclerotia,
mycelia, or filaments. For example, the sclerotia of ergot have been found to
contain as much as 60 per cent of oil. In higher plants, many seeds contain
high percentages of oil, so as to make them commercial sources for edible or
lubricating oils, such as olive oil, rape-seed oil, cottonseed oil, castor oil,
corn oil, sunflower-seed oil, etc., etc. Nuts often contain large proportions of
oil, the kernel of the Brazil nut, for example, sometimes contains as high as
70 per cent of oil, while an oil content of 50 per cent, or more, is common in
almonds, walnuts, etc.

Oils also occur as reserve food material in other storage organs of plants,
such as the tubers of certain flowering plants, and the roots of many species
of orchids. Sometimes the appearance of oils in the stems of trees, or the
winter leaves of evergreens, seems to be only temporary and to occur only
during periods of very low temperatures.

Much less frequently, fats or oils are found in the vegetative organs of
plants, as in the leaves of evergreens. Their appearance and functions in
these organs seem to be much less certain than in the other cases cited
above; although in rare cases a considerable proportion of oily material has
been found to exist in definite association with the chloroplasts.

The vegetable fats and oils have many important industrial uses. Some of
them, such as olive oil, cottonseed oil, cocoanut oil, etc., are largely used as
human food. Others, as castor oil, are used as lubricants. The so-called
"drying oils" (see page 132), such as linseed oil, etc., are used in the
manufacture of paints and varnishes. Some cheap vegetable oils are used as
the basis for the manufacture of soaps, etc. Hence, industrial plants and
processes for the extraction of oils from plant tissues are of very great
economic importance.

CHEMICAL CONSTITUTION

The fats (of either plant or animal origin) are glycerides, i.e., glycerol
esters of organic acids. As has been pointed out, esters are derived from
organic acids and alcohols in exactly the same way that mineral salts are
derived from inorganic acids and metallic bases.

Glycerol is, however, a trihydric alcohol, i.e., it contains three replaceable
(OH) groups. Its formula is C3H5(OH)3, or CH2OH·CHOH·CH2OH. Hence,
three molecules of a monobasic acid are required to replace all of its (OH)
groups.

For example,

It is theoretically possible, of course, to replace either one, two, or three of
the (OH) groups in the glycerol with acid radicals, thus producing either
mono-, di-, or triglycerides. If the primary alcohol groups in the glycerine
molecule are designated by (1)
and the secondary one by (2), thus, CH2(1)OH·CH(2)OH·CH2(3)OH, it is
conceivable that there may be either (1) or (2) monoglycerides, either (1, 1)
or (1, 2) diglycerides, or a triglyceride, depending upon which of the (OH)
groups are replaced. Compounds of all of these types have been produced by

combinations of glycerol with varying proportions of organic acids under
carefully controlled conditions; and all of them found to possess fat-like
properties. All natural fats are triglycerides, however. Most natural fats are
mixtures of several different triglycerides in each of which the three (OH)
groups of the glycerol has been replaced by the same organic acid radical, as
in the example of stearin shown above. But recent investigations have shown
that some of the common animal fats, and perhaps some plant oils, may be
made up of mixed glycerides, i.e., those in which the different (OH) groups
have been replaced by different acid groups, as oleo-stearin, oleo-stearo-
palmitin, etc.

THE ACIDS WHICH OCCUR IN NATURAL FATS

The acids which, when combined with glycerol, produce fats are of two
general types. The first of these are the so-called "fatty acids" having the
general formula CnH2n+{1}·COOH. These are the "saturated" acids, i.e.,
they contain only single-bond linkages in the radical which is united to the
·COOH group; hence, they cannot take up hydrogen, oxygen, etc., by direct
addition. The second type are the "unsaturated" acids belonging to several
different groups, as discussed below, but all having one or more double-
linkages between the carbon atoms of the alkyl radical which they contain.
Because of these double linkages, they are all able to take on oxygen,
hydrogen, or the halogen elements, by direct addition. When exposed to the
air, for example, these "unsaturated" acids, or the oils derived from them,
take up oxygen, increasing in weight, and becoming solid or hard and stiff.
Hence, natural oils which contain considerable proportions of glycerides of
these "unsaturated" acids are known as "drying oils" and are largely used in
the manufacture of paints, varnishes, linoleums, etc.; while oils which
contain little of these glycerides are known as "non-drying," and are used for

food, for lubrication, or for other technical purposes in which it is essential
that they remain in unchanged fluid condition when exposed to the air.

The following are some of the more important of the acids which occur as
glycerides in natural fats: Saturated Acids:

(a) Acetic, or stearic, acid series—general formula, CnH2n+1·COOH.
(1) Formic acid, H·COOH, occurs free in nettles, ants, etc.
(2) Acetic acid, CH3·COOH, occurs free in vinegar.
(3) Butyric acid, C3H7·COOH, in butter fat.
(4) Capric acid, C9H19·COOH, in butter fat and cocoanut oil.
(5) Myristic acid, C13H27·COOH, in cocoanut oil and spermaceti.
(6) Palmitic acid, C15H31·COOH, in palm oil and many fats.
(7) Stearic acid, C17H35·COOH, in most fats and oils.

Intervening members of this series, such as caprylic acid, C7H15·COOH,
and lauric acid, C11H23·COOH, are also found in smaller quantities in
cocoanut and palm nut oils, in butter fat, and in spermaceti; while higher
members of the series, as arachidic acid, C19H39·COOH, and lignoceric acid,
C23H47·COOH, are found in peanut oil; and cerotic acid, C25H51·COOH,
and melissic acid, C29H59·COOH, in beeswax and carnauba wax.
Unsaturated Acids:

(b) Oleic acid series—general formula, CnH2n-1·COOH.
(1) Crotonic acid, C3H5·COOH, occurs in croton oil.
(2) Oleic acid, C17H33·COOH, occurs in many fats and oils.
(3) Brassic acid, C21H41·COOH, occurs in rape-seed oil.
(4) Ricinoleic acid, C17H32OH·COOH, occurs in castor oil.
(c) Linoleic acid series—general formula, CnH2n-3·COOH.
(1) Linoleic acid, C17H31·COOH, occurs in linseed and other
drying oils.
(d) Linolenic acid series—general formula, CnH2n-5·COOH.

(1) Linolenic acid, C17H29·COOH, occurs in many drying oils.

It will be observed that all of these acids contain a multiple of two total
carbon atoms. No acid containing an uneven number of carbon atoms has
been found in a natural fat. Furthermore, the acids which occur most
commonly in natural fats are those which contain eighteen carbon atoms; in
fact, more than 80 per cent of the glycerides which compose all animal and
vegetable fats are those of the C18 acids. This fact, in addition to the one that
the sugars and starches all contain multiples of six carbon atoms in their
molecules, indicates a very great biological significance of the chain of six
carbon atoms. This has been alluded to in connection with the discussion of
the biological significance of molecular configuration (see page 57) and will
be mentioned again in other connections.

THE ALCOHOLS WHICH OCCUR IN NATURAL
FATS

Glycerol, as has been pointed out, is by far the most common alcoholic
constituent of natural fats and oils. This substance, which is familiar to
everyone under its common name "glycerine," is a colorless, viscid liquid
having a sweetish taste. It is a very heavy liquid (specific gravity 1.27)
which mixes with water in all proportions and when in concentrated form is
very hygroscopic.

Glycerine is made from fats and oils by commercial processes which
clearly prove that the constitution of fats is as described above. The fat is
boiled with a solution of caustic soda and is decomposed, the sodium of the
alkali taking the place of the glyceryl (C3H5) group, the latter combining
with three (OH) groups from the three molecules of alkali necessary to
decompose the fat. A sodium salt of the organic acid, or soap, and glycerol

are thus produced, and are separated by saturating the hot solution with
common salt, which causes the soap to separate out as a layer on the surface
of the liquid, which, on cooling, solidifies into a solid cake, which is then cut
and pressed into the familiar bars of commercial soap. From the remaining
solution, the glycerine is recovered by evaporation and distillation under
reduced pressure. Taking stearin, a common fat, as the example, the reaction
which takes place in the above process may be expressed by the following
equation:

C3H5(C17H35·COO)3 + 3NaOH = 3C17H35COONa + C3H5(OH)3
Stearin Sodium stearate—a soap Glycerol

This process, since it yields soap as one of its products, is called
"saponification." All fats, when saponified, yield soaps and either glycerol or
(more rarely) some of the other alcohols which are described below.

Glycerine is also prepared from fats by hydrolysis with superheated
steam. Using olein, a glyceride which is present in olive oil and many
common fats, as the example in this case, the equation for the reaction is:

C3H5(C17H33·COO)3 + 3H2O = 3C17H33·COOH + C3H5(OH)3
Olein Steam Oleic acid Glycerol

In this case the free fatty acid, instead of a soap, is the product which is
obtained in addition to glycerol.

In the equations presented above, a single glyceride has been used as the
example in each case. In the saponification, or hydrolysis, of natural fats and
oils which, as has been shown, are mixtures of many glycerides, the resultant
soaps, or fatty acids, are mixtures of as many compounds as there were
individual glycerides of the original fat, but the glycerol is identical in every
case.

When glycerol is heated with dehydrating agents, it is easily converted
into acrolein, an unsaturated aldehyde having a peculiar characteristic
pungent odor. Hence, the presence of glycerol, or glycerides, in any
substance may usually be detected by mixing the material with anhydrous
acid potassium sulfate and heating the mixture in a test tube, when the
characteristic odor of acrolein will appear.

Glycerol possesses all the characteristic properties of an alcohol, forming
alcoholates with alkalies, esters with acids, etc. It is an active reducing agent,
being itself easily oxidized to a variety of different products depending upon
the strength of the oxidizing agent used and the conditions of the
experiment. Microorganisms affect it in a variety of ways, either converting
it into simple fatty acids, or condensing it into longer-chain compounds.

Open-chain monohydric alcohols, higher members of the ethyl alcohol
series, such as cetyl, C16H33OH, carnaubyl, C24H49OH, ceryl, C26H53OH,
and melissyl, C30H61OH, are found in the esters which constitute the major
proportion of the common waxes.

Cholesterol and phytosterol are empirical names for certain closed-ring,
monohydric alcohols which are found in relatively small amounts in all fats,
the former term designating those found in animal fats and the latter those of
plant origin. Their composition has not yet been definitely established. They
are known to contain two, or three, closed rings, probably of the
phenanthrene type; to form dichlor- and dibrom- addition products, showing
that they contain one side-chain double linkage; and to yield ketones when
oxidized, indicating that they are secondary alcohols. They form acetyl
esters, or acetates, which can be separated from each other and identified by
their crystal forms and melting points. Because of this fact and of the further
fact that they are present in detectable quantities in practically all fats and
oils, they afford a qualitative means of distinguishing between fats of animal

and of plant origin. This possibility is the most interesting fact known
concerning these complex alcohols; although their presence as esters in all
plant and animal fats indicates that they must have some biological function.

Phytosterol is not a single alcohol, but a mixture of at least two, which
have been separated and studied as sitosterol, C27H43OH, and stigmasterol,
C30H49OH. As has been said, these are found in small proportions in all
vegetable fats, being present in largest amounts in oily seeds, especially
those of the legumes.

The saponification of esters of cholesterol and phytosterol is a difficult
and unsatisfactory process; but since this affords the only known means to
distinguish between fats of plants and of animal origin, its technique has
been fairly well worked out, and the process used in the study of the changes
which take place in plant fats when they are used by animals as food.

HYDROLYSIS AND SYNTHESIS OF FATS

The reaction for the hydrolysis of fats has been discussed in connection
with the process for the manufacture of glycerine. This reaction takes place
very slowly with cold water alone, can be easily brought about by the action
of superheated steam, and much more easily and rapidly in the presence of
some catalyst (sulfuric acid is an especially effective catalyst for this
purpose).

Fats can be artificially synthetized by heating mixtures of glycerol and
fatty acids, under considerable pressure, for some time at temperatures of
200° to 240° C.; or by heating a mixture of the disulfuric ester of glycerol
with a fatty acid dissolved in sulfuric acid. Recently, fatty acids have been
prepared from carbohydrates, by first breaking the hexoses down into three-

carbon compounds, then carefully oxidizing these to pyruvic acid,
CH3·CO·COOH, which can then be condensed into acids having longer
chains. The violent reagents and long-continued processes which must be
employed for the artificial hydrolysis or synthesis of the fats are in sharp
contrast with the easy and rapid transition of carbohydrates to fats, and vice
versa, which take place in both plant and animal nutrition.

THE EXTRACTION OF OILS FROM PLANT
TISSUES

There are three types of methods which are employed for the extraction of
oil from oil-bearing seeds, etc., either as a commercial industry or for the
purposes of scientific study. These are (1) by pressure; (2) extraction with
volatile solvents; and (3) boiling the crushed seeds or fruits with water.

By the first method, the seeds are first cleaned, then "decorticated" (hulls
removed), crushed or ground, then subjected to intense pressure in an
hydraulic press. In the commercial process, the ground seeds are first
pressed at ordinary temperature, which yields "cold-drawn" oil, then the
press cake is heated and pressed again, whereby "hot-drawn" oil is obtained.
The crude oil is refined by heating it to coagulate any albumin which it may
contain, and is sometimes bleached by different processes before it is
marketed. The press cake from many seeds, such as flaxseed (linseed),
cottonseed, etc., is ground up and sold for use as stock feed.

In the second method, the finely crushed seeds are treated with solvents
such as gasoline or carbon bisulfide, in an apparatus which is so arranged
that the fresh material is treated first with solvent which has already passed
through various successive lots of material and has become highly charged
with the oil, followed by other portions which contain less oil, and finally by

fresh solvent, whereby the last traces of oil are removed from the material.
The saturated solvent is transferred to suitable boilers and the solvent
distilled off and condensed for repeated use, leaving the oil in the boiler in
very pure form.

Extraction by boiling with water is sometimes used in the preparation of
castor oil and olive oil. In such cases, the crushed seeds are boiled with
water and the oil skimmed off as fast as it rises to the surface.

IDENTIFICATION OF FATS AND OILS

Fats and oils are identified by determinations of their physical properties,
such as specific gravity, melting point, refractive index, etc., and by certain
special color reactions for particular oils; or by measurements of certain
chemical constants, such as the percentage of free fatty acids which they
contain, the saponification value (i.e., the number of milligrams of KOH
required to completely saponify one gram of the fat), the iodine number
(percentage by weight of iodine which is absorbed by the unsaturated fatty
acids present in the fat), percentage of water-insoluble fatty acids obtained
after saponification and acidifying the resultant soap, etc., etc. Most of these
tests must be carried out under carefully controlled conditions in order to
insure reliable identifications, and need not be discussed in detail here. Full
directions for making such tests, together with tables of standard values for
all common fats and oils, may be found in any reference book on oil
analysis.

PHYSIOLOGICAL USE OF FATS AND OILS

In animal organisms, fats are the one important form of energy storage.
They also form one of the most important supplies of energy reserve
material in plants. Carbohydrates commonly serve this purpose in those
plants whose storage reservoirs are in the stems, tubers, etc.; but in most
small seeds the reserve supply of energy is largely in the form of oil, and
even in those seeds which have large endosperm storage of starch, the
embryo is always supplied with oil which seems to furnish the energy
necessary for the first germinative processes.

Fats are the most concentrated form of potential energy of all the different
types of organic compounds which are elaborated by plants. This is because
they contain more carbon and hydrogen and less oxygen in the molecule
than any other group of substances of vegetable (or animal) origin. It has
been pointed out that a quantity of fat capable of yielding 100 large calories
of heat will occupy only about 12 cc. of space, whereas from 125 to 225 cc.
of space in the same tissue would be required for the amount of starch of
glycogen necessary to yield the same amount of heat, or energy, when
oxidized.

The fats undoubtedly catabolize first by hydrolysis into glycerol and fatty
acids, and then by oxidation possibly first into carbohydrates and then finally
into the end-products of oxidation, namely, carbon dioxide and water. The
following hypothetical equation to represent the oxidation of oleic acid into
starch, suggested by Detmer, is interesting as a suggestion of how much
oxygen is required and how much heat would be liberated by such a
transformation:

C18H34O2 + 27O = 2(C6H10O5) + 6CO2 + 7H2O

Complete oxidation of oleic acid to the final end-products, carbon dioxide
and water, would require much more oxygen, thus:

C18H34O2 + 51O = 18CO2 + 17H2O.

Hence, Detmer's reaction would yield only approximately one-half the
total energy available in the acid; but it does indicate the possibility of
redevelopment of fatty acids or fats from the unoxidized carbohydrate
material which remains in the equation. Moreover, there is abundant
evidence to show that, in both animal and plant tissues, energy changes are
brought about chiefly by the transformation of fats into carbohydrates and
vice versa.

Many different hypotheses have been put forward concerning the mode of
transformation of fats into carbohydrates, and the changes which take place
in oily seeds during their germination have been carefully studied by many
investigators. The following seem to be fairly well established facts. First,
that fats as such may be translocated from cell to cell, since cell-walls and
cell protoplasm seem to be permeable to oil if it is a sufficiently fine
emulsion; or they may be hydrolyzed into glycerol and fatty acids and
translocated from cell to cell in these forms and recombined into fats in the
new location. Second, that fats are formed from glucose in some plants, from
sucrose and from starch in others, and from mannite and similar compounds
in still other species. Third, that in germination the fatty acids are used up in
the order of their degree of unsaturation, those which contain the largest
number of double-bond linkages being used first, and the saturated acids last
of all. Fourth, that the sugar produced by the oxidation of fats is derived
either from the glycerol or from the fatty acids of the fat, depending upon the
nature of the latter. If the fat is saturated, the glycerine is converted into
sugar while the fatty acids are oxidized; but if the fat contains large
proportions of unsaturated acids, these contribute to the formation of sugar.

Recent studies seem to show that in the animal body fats serve an
important function in connection with the production of antibodies to disease
germs. But there is as yet no evidence to show that fats and oils have any

similar function in plant tissues. The fact that they are found almost wholly
in the storage organs of plants seems to indicate that their use as food reserve
material is their principal, if not their sole, function in the plant economy.

B. THE WAXES

Waxes are most commonly found in or on the skin of leaves or fruits.
They are similar to fats in chemical composition, except that, instead of
being glycerides, they are esters of monohydric alcohols of high atomic
weight. The term wax, when used in the chemical sense, has reference to this
particular type of esters rather than to any special physical properties which
the compound possesses, and both solid and liquid waxes are known.

Carnauba wax, found on the leaves of the wax-palm (Copernicia cerifera)
contains ceryl alcohol (C23H53OH) and myricyl alcohol (C30H61OH) esters
of cerotic acid (C25H51·COOH) and carnaubic acid (C23H47·COOH). It is
the best known vegetable wax. Poppy wax is composed chiefly of the ceryl
ester of palmitic acid (C17H35·COOH).

Since waxes contain no glycerol, they give no odor of acrolein when
heated with dehydrating agents, do not become rancid, and are less easily
hydrolyzed than the fats. They are soluble in the same solvents as the fats,
but generally to a less degree.

The facts that waxes are impervious to water and usually occur on the
surfaces of plant tissues have led to the conclusion that their chief function is
to provide against the too-rapid loss of water by evaporation from these
tissues. This seems to be borne out by the common experience that many
fresh fruits and vegetables will keep longer without shriveling if their waxy

coating is undisturbed. No other function than that of regulation of water
losses has been suggested for the plant waxes.

C. THE LIPOIDS

The lipoids, or "lipins," as some authors prefer to call them, are
substances of a fat-like nature which are found in small quantities in nearly
all plant and animal tissues and in considerable proportions in nerve and
brain substance, in egg yolk, etc., and in the seeds of plants. When
hydrolyzed, they yield fatty acids or derivatives of fatty acids and some
other group containing either nitrogen only or both nitrogen and phosphorus.
The facts that they are extracted from tissues by the same solvents which
extract fats and that they yield fatty acids when hydrolyzed account for the
name "lipoid," which comes from the Greek word meaning fat. Some
writers, who object to the word "lipoid" as a group name, prefer to call these
substances the "fat-like bodies."

The first group of lipoids to be studied were those which occur in the
brain; and the name cerebroside was given to those lipoids which, when
hydrolyzed, yield fatty acids, a carbohydrate and a nitrogen-containing
compound but no phosphoric acid; while those lipoids which contain both
nitrogen and phosphorus were called phosphatides. Substances which
correspond in composition to both these types are found in plant tissues and
the same class names are applied in a general way to lipoids of either plant
or animal origin.

Plant lipoids have not been studied to nearly the same extent as have those
which occur in the animal body; and certain observers believe that there are
significant differences between the lipoids of plants and those of animal
origin. However, most investigators use the same methods of study and the

same systems of nomenclature for these fat-like substances, regardless of
their origin.

LECITHIN

This phosphatide is by far the best-known lipoid. It occurs in the brain, the
heart, the liver, and in the yolk of the eggs of many animals; and either
lecithin or a substance so nearly like it in character as to be regarded by most
investigators as identical with it, is present in small, but constant, quantities
in nearly all seeds, especially those of leguminous plants. In many legume
seeds, it constitutes from 50 to 60 per cent of the "ether extract," or "crude
fat," which can be extracted from the crushed seeds, using ether as the
solvent.

Lecithin is a glyceride. Only two of the (OH) groups of the glycerol are
replaced by fatty acids, however; the third being replaced by phosphoric
acid, H3PO4, or PO(OH)3, which, in turn, has one of its hydrogen atoms
replaced by the base choline. Choline is a nitrogenous base, or amine, which
may be regarded as ammonium hydroxide with three of its hydrogen atoms
replaced by methyl groups and the fourth by the ethoxyl group, the latter
being the ethyl group with an OH in place of one of its hydrogens. Thus,

Without the choline, lecithin would be a di-fatty acid derivative of
glycero-phosphoric acid. These relations may be seen in the following
formulas:

There are many different possible linkages of the constituent groups
which make up the lecithin molecule. In the first place, if the (OH) groups of
the glycerol molecule be numbered (1) and (2), thus,

the fatty acid radicals may be attached either in one (1) position and one
(2) position, or in the two (1) positions; hence, two forms of glycero-
phosphoric acid are possible, thus

Again, the choline may be attached to the phosphoric acid either through
its alcoholic (OH) group or through its basic (N) group, thus

The facts that in the arrangement (B) the central carbon atom of the
glycerol would be asymmetric, and that both lecithin and the glycero-
phosphoric acid derived from it by hydrolysis are optically active, prove that
formula (B) correctly represents the arrangement of that part of the lecithin
molecule; and there is ample theoretical and experimental evidence to prove
that the choline linkage is through the alcoholic (OH) group. Hence the
formula for lecithin indicating the linkage as shown above is the correct one.

The fatty acids in the lecithin molecule may be different in lecithins from
different sources, just as they are different in fats from different sources.
Both oleic acid and a solid fatty acid have been found in the hydrolysis
products of lecithin from leguminous seeds. In certain lupine seeds, the fatty
acids present in the lecithin appear to be palmitic and stearic.

OTHER PLANT PHOSPHATIDES

Phosphatides other than lecithin are common in plants. In these, various
sugars replace part or all of the glycerol as the alcoholic part of the ester.
Percentages of sugar varying from mere traces up to 17 per cent of the
weight of material taken, have been found in the products of hydrolysis of
phosphatides prepared from vetch seeds, potato tubers, plant pollens, and
whole wheat meal.

Furthermore, betaine

and perhaps other vegetable amines (see Chapter XII) sometimes replace
choline as the basic group in the phosphatides.

PLANT CEREBROSIDES

Bodies similar to the animal cerebrosides seem to occur in many plant
tissues, since plant lipoids which yield no phosphorus when hydrolyzed have
often been isolated. The sugar which constitutes the alcoholic portion of
their structure appears to be galactose in every case which has been reported.
Beyond this, little is known of the structure of these plant cerebrosides, as
they are very difficult to prepare in pure form and not easily hydrolyzed.

PHYSIOLOGICAL USES OF LIPOIDS

Lipoids are so universally present in plant and animal tissues and so
commonly found in those parts of the organism in which vital phenomena
are most pronounced (brain, heart, embryo of egg, embryo of seeds, etc.),
that it is evident that they must play some important rôle in the activity of
living protoplasm. There is, as yet, however, no definite and certain
knowledge of what this rôle is. Various theories concerning the matter have
been put forward in recent years. For example, Overton, in 1901, presented
the idea that every living cell is surrounded by a semi-permeable membrane

consisting of lipoid material, which regulates the passage into and out of the
cell of substances necessary to its metabolism and growth. Recent
investigations by Osterhout and others indicate, however, that Overton's
hypothetical lipoid membrane is not essential to a proper explanation of the
migration into and out of the cell protoplasm of nutritive materials, etc.
Other investigators have cited results which appear to indicate that lipoids
play an important, but as yet unknown, part in the process of fat metabolism.
Others go even further than this, and argue that since the extraordinary
rapidity of the chemical changes which take place in plant protoplasm
indicates the necessity of the presence there of exceedingly labile
substances, and since both fats and proteins are relatively stable compounds,
it is possible that the lipoids, which contain both nitrogenous and fatty acid
groups, play an exceedingly important part in the metabolism processes.
Bang, in particular, has pointed out (in 1911) that the lipoids are probably
the most labile of all the components which constitute the colloidal system
known as plant protoplasm. The importance of such considerations will be
more apparent after the relation of colloidal phenomena to the activities of
plant cell contents has been more fully discussed (see Chapter XVI).

Experimental studies of the physiological uses of lipoids have thus far
been devoted almost exclusively to those of animal tissues. They have been
seriously hampered by the difficulty of securing properly purified extracts of
lecithin and similar lipoids. The same labile character which apparently
makes them so important in the chemical changes in the cell makes them
equally unstable compounds to work with in attempting to secure pure
preparations for the purposes of experimental study. On this account, there
is, as yet, no certain knowledge concerning their actual physiological uses. It
is evident, however, that they have some really important rôle to play, which
opens up a promising field for further study.

References.

Abderhalden, E.—"Biochemisches Handlexikon, Band 3, Fette,
Wachse, Phosphatide, Cerebroside, ..." 340 pages, Berlin, 1911.

Hopkins, E.—"The Oil-Chemist's Handbook," 72 pages, New
York, 1902.

Leathes, J. B.—"The Fats," 138 pages, Monographs on
Biochemistry, London, 1913.

Lewkowitsch, J.—"Chemical Technology and Analysis of Oils,
Fats, and Waxes," Vol. I, 542 pages, 54 figs.; Vol. II, 816 pages,
20 figs.; and Vol. III, 406 pages, 28 figs., London, 1909.

Maclean, H.—"Lecithin and Allied Substances," 206 pages,
Monographs on Biochemistry, London, 1913.

Southcombe, J. E.—"Chemistry of the Oil Industries," 204
pages, 13 figs., London, 1918.

CHAPTER XI

ESSENTIAL OILS AND RESINS

Included in this group are all those substances to which the characteristic
odors of plants are due, along with others similar in structure and possessing
characteristic resinous properties. They have no such uniformity in
composition as is exhibited by the oils which are included among the fats
and waxes; but belong to several widely different chemical groups.
Furthermore, there is no sharp dividing line between the essential oils and
certain esters of organic acids on the one hand and the fats on the other. For
example, if an aromatic fluid essence is a light fluid, non-viscid, and easily
volatile, it is usually classed with the organic esters; denser liquid
substances, of oily or waxy consistency, and with comparatively slight odor
and taste are usually fats, while oils of similar physical properties but
possessing strong characteristic odors are classed as essential oils, regardless
of their chemical composition.

Included in this general class are compounds having a great variety of
chemical structures; e.g., hydrocarbons, alcohols, phenols, organic sulfides
and sulfocyanides, etc. Many of these compounds are crystalline solids at
ordinary temperatures, but melt to oily fluids at higher temperatures. The
characteristic property which assigns any given plant extract to this group is

that it has a strikingly characteristic odor or taste, often accompanied by
some definite physiological effect, or medicinal property.

These compounds may be either secretions or excretions of plants,
sometimes normally present in the healthy tissue, and sometimes produced
as the result of injury or disease.

The essential oils and the resins often occur associated together in the
plant; or, the resins may develop from the oily juice of the plant after
exposure to the air.

THE ESSENTIAL OILS

These may be divided, according to their chemical composition, into two
major groups; (1) the hydrocarbon oils, or terpenes, and (2) the oxygenated
and sulfuretted oils.

The terpenes are of three different types, namely: (a) the hemiterpenes,
C5H8, unsaturated compounds of the valerylene series, of which isoprene
(found in crude rubber) is the best-known example; (b) the terpenes proper,
C10H16, which constitute the major proportion of the whole group; and (c)
the polyterpenes (C5H8)n, of which colophene and caoutchouc are the most
common examples.

Eleven different terpenes having the formula C10H16 have been isolated
from various plant juices, and their molecular arrangement carefully worked
out. The following three examples will serve as typical of the general
structural arrangement of these hydrocarbons:

A discussion of the evidence which supports these formulas as properly
represented the molecular arrangements of the various isomeric forms would
be out of place here, as its only particular interest is in connection with the
medicinal effects of the different compounds. It is clear, however, that they
are six-membered hydrocarbon rings, with additional hydrocarbon groups
attached to one or more of the carbon atoms in the ring.

Different modifications, or varieties, of the terpenes constitute the main
proportions of the oils of turpentine, bergamot, lemon, fir needles,
eucalyptus, fennel, pennyroyal, etc.

The oxygenated essential oils may be either alcohols, aldehydes, ketones,
acids, esters, or phenols, derived from either five-membered or six-
membered closed-ring hydrocarbons. They are usually present in the plant
oil in mixtures with each other or with a terpene. Since most of them have
pronounced physiological or medicinal properties, their structure has been
well worked out, in most cases; but it seems to be hardly worth while to
present these matters in detail here, as they are of interest chiefly on account
of their medicinal properties rather than their botanical functions.

Borneol, C10H17OH, and menthol, C10H19OH, are typical alcohols. The
latter is a crystalline substance, which melts at 42°, which is present in
peppermint oil, both as the free alcohol and as an ester of acetic acid.

Amyl acetate, CH3·COOC5H11, and linalyl acetate, CH3·COOC10H17, the
latter occurring in the oils of lavender and bergamot, are typical esters
classed as essential oils.

As examples of the aldehyde oils, benzoic aldehyde, C6H5CHO, "oil of
bitter almonds," and cinnamic aldehyde, C6H5CH=CHCHO, found in the
oils of cinnamon and cassia, may be cited.

Camphor, C10H16O, is a ketone, having the following structural formula:

There are a considerable number of essential oils which are phenols.
Thymol, C6H3·(CH3)·(C3H7)·OH, in oil of thyme, and carvacrol, its isomer,
in oil of hops, are familiar examples.

Coumarin, the anhydride of cinnamic acid, ; is an
example of an acid substance which is classed as an essential oil, even
though it is a solid at ordinary temperatures. It has an odor and flavor similar

to that of vanillin, the essential flavoring material of the vanilla bean, and is
often used as a substitute for the latter in the preparation of artificial
flavoring extracts.

Of the essential oils containing sulfur, there are two common examples;
oil of mustard, allyl isosulfocyanide, C3H5NCS, and oil of garlic, allyl
sulfide (C3H5)2S. The latter is present in onions, garlic, water cress, radishes,
etc., the difference in flavor of these vegetables being due to the fact that the
allyl sulfide is united with other different groups in the glucoside
arrangement, in the different plants. Similarly, mustard oil is not present in
mustard seeds as such, but as a glucoside which, when hydrolyzed by the
enzyme myrosin which is always present in other cells of the same seeds,
yields C3H5NCS, KHSO4, and C6H12O6.

THE RESINS

The resins were formerly supposed to be the mother substances from
which the terpenes are derived. It is now known, however, that they are the
oxidation products of the terpenes. Their exact structure is still a matter of
some uncertainty, as their peculiar "resinous" character makes them very
difficult to study by the usual methods of chemical investigations.

Resins are divided into two classes: (a) the balsams, and (b) the solid or
hard resins. Canada balsam and crude turpentine are familiar examples of the
first class. They consist of resinous substances, dissolved in or mixed with
fluid terpenes. Ordinary resin, or colophony, consists chiefly of a monobasic
acid having the empirical formula C20H30O2, known as sylvinic acid, whose
exact structure is not known. Its sodium salt is used as the basis for cheap
soaps.

The hard resins are amorphous substances of vitreous character, which
consist of very complex aromatic acids, alcohols, or esters, combined with
other complicated structures, known as resenes, whose definite chemical
nature is not yet known. Among the hard resins are many substances which
are extensively used in the manufacture of varnishes, such as copal, amber,
dammar, sandarach, etc.

There are also resinous substances, such as asafœtida, myrrh, gamboge,
etc., which are mixtures of gums (see Chapter VI) and true resins. Some of
these have considerable commercial value for medicinal or technical uses.

PHYSIOLOGICAL USES AND BIOLOGICAL
SIGNIFICANCE OF ESSENTIAL OILS

No theory has yet been advanced concerning the possibility of the use of
essential oils and resins by plants in their normal metabolic processes. The
very great diversity in their chemical nature makes it impossible that they
should all be considered as having the same physiological function, if indeed
any of them actually have any such function.

It is evident that those aromatic compounds which occur as normal
secretions of plants and which give to the plants their characteristic odors
may act either as an attraction to animals which might utilize the plants as
food and so serve to distribute the seed forms, or as a repellent to prevent the
too rapid destruction of the leaves, stems, or seeds of certain species of
plants whose slow-growing habits require the long-continued growth of
these portions of the plant for the perpetuation of the species. The presence
of these compounds in larger proportions in those species of conifers, etc.,
which grow in tropical regions, in competition with other rapid-growing
vegetation, suggests the latter possibility. It must be admitted, however, that

their presence in such cases may be the result of climatic conditions, as
indicated by the fact that most spice plants are tropical in habit, rather than
the result of their protective influence in the struggle for survival during past
ages.

Many of the oils and resins which are secreted as the result of injury by
disease or wounds have marked antiseptic properties and undoubtedly serve
to prevent the entrance into the injured tissue of destructive organisms.

But apart from these possible protective influences which may have had
an important effect upon the preservation and perpetuation of the species of
plants which secrete them, there is no known biological necessity for the
presence of these aromatic substances in plants.

References.

Abderhalden, E.—"Biochemisches Handlexikon, Band 7,
Gerbstoffe, Flechtenstoffe, Saponine, Bitterstoffe, Terpene,
Aetherische Oele, Harze, Kautschuk," 822 pages, Berlin, 1912.

Allen's Commercial Organic Analysis, Vol. IV, "Resins, Rubber,
Guttapercha, and Essential Oils," 461 pages, 7 figs.,
Philadelphia, 1911 (4th ed.).

Heusler, F., trans. by Pond, F. J.—"The Chemistry of the
Terpenes," 457 pages, Philadelphia, 1902.

Parry, E. J.—"The Chemistry of Essential Oils and Perfumes,"
401 pages, 20 figs., London, 1899.

CHAPTER XII

THE VEGETABLE BASES

We come, now, to the consideration of the characteristically nitrogenous
compounds of plants. None of the groups of compounds which have been
considered thus far have, as a group, contained the element nitrogen. This
element is present in the chlorophylls and in certain other pigments, but not
as the characteristic constituent of the molecular structure of the group of
compounds, nor do these compounds serve as the source of supply of
nitrogen for the plant's needs.

The characteristic nitrogen-containing compounds may all be regarded as
derived from ammonia, or ammonium hydroxide, by the replacement of one
or more hydrogen atoms with organic radicals of varying type and
complexity. If the group, or groups, which be considered as having replaced
a hydrogen atom in ammonia, in such compounds, is an alkyl group, the
compound is strongly basic in character and is known as an amine; whereas
if the replacing group is an acid radical, the resulting compound may be
neutral (known as acid amides), or weakly acid (known as amino-acids) in
type. Compounds of the first type constitute the vegetable bases; while those
of the second type are the proteins.

The vegetable bases may be divided into three groups. These are (a) the
plant amines, which are simple open-chain amines; (b) the alkaloids, which
are comparatively simple closed-ring amines, containing only one nitrogen
atom in any single ring; and (c) the purine bases, which are complex
compounds containing a nucleus with four carbon atoms and four nitrogen
atoms arranged alternately to form a double-ring group.

THE PLANT AMINES

The simple amines bear the relation to ammonia, or ammonium
hydroxide, represented by the following formulas, in which the R indicates
any simple alkyl radical:

The simple amines which occur in animal tissues are known as
"ptomaines" and "leucomaines." The ptomaines are all decomposition
products resulting from the putrefactive decay of proteins caused by moulds
or bacteria. Some of these are highly toxic, producing the so-called
"ptomaine-poisoning"; while others are wholly innocuous. They are all
simple amines. Putrescine, di-amino butane, NH2·CH2·CH2·CH2·CH2·NH2,
and cadaverine, di-amino pentane, HN2·(CH2)5·NH2, are common non-toxic
ptomaines, resulting from the decay of meat. Neurine, trimethyl-ethylene
ammonium hydroxide, (CH3)3(C2H3)·NOH, is a violently poisonous
ptomaine produced in the decay of fish. Amines of similar structure to these
are occasionally found in living animal tissues. Such compounds are known

as leucomaines, to distinguish them from the ptomaines, which are found
only in dead material.

Corresponding in structure and properties to these amines of animal
origin, there is a series of basic substances, found in many plants, known as
the plant amines. The following are common examples:

Trimethyl amine, (CH3)3N, is a very volatile compound, found in the
flowers of several species of the Rose family, the leaves of certain weeds,
etc. When crushed, these tissues give off a very fetid odor, which is due to
this amine.

Choline, muscarine, and betaine are plant amines which are closely
related to each other and to neurine (the toxic ptomaine) in composition and
structure, as shown in the following formulas:

Choline and betaine are non-toxic; while muscarine and neurine are
violent poisons.

Choline and muscarine occur in certain toadstools. Betaine and choline
often occur together in the germs of many plants. Betaine is found in the
beet root and the tubers of Jerusalem artichoke. Choline occurs alone in the

seeds and fruits of many plants, sometimes as the free amine, but more often
as a constituent of lecithin (see page 141).

Phenyl derivatives of simple amines are sometimes found in plants.
Hydroxyphenylethyl amine,

found in ergot, and hordein,

found in barley, are examples. The former has marked medicinal
properties.

There is no known physiological use for these simple amines in plants. By
some investigators, they are regarded as intermediate products in the
synthesis or decomposition of proteins; but it would seem that if this were a
normal procedure, these amines would occur in varying proportions in all
plants, under different conditions of metabolism, instead of in practically
constant proportions in only a few species, as they do.

ALKALOIDS

These are a group of strong vegetable bases whose nitrogen atom is a part
of a closed-ring arrangement.

As a rule, alkaloids are colorless, crystalline solids, although a few are
liquids at ordinary temperatures. They are generally insoluble in water, but
easily soluble in organic solvents. Being strong bases, they readily form salts
with acids, and these salts are usually readily soluble in water.

Alkaloids are usually odorless; although nicotine, coniine, and a few
others, have strong, characteristic odors. Most of them have a bitter taste,
and many of them have marked physiological effects upon animal
organisms, so that they are extensively used as narcotics, stimulants, or for
other medicinal purposes.

Most of the alkaloids contain asymmetric carbon atoms and are, therefore,
optically active, usually levorotatory, although a few are dextrorotatory.

The alkaloids are precipitated out of their solutions by various solutions of
chemical compounds, known as the "alkaloidal reagents": iodine dissolved
in potassium iodide solution gives a chocolate-brown precipitate; tannic
acid, phosphotungstic acid, phosphomolybdic acid, and mercuric iodide
solutions give colorless, amorphous precipitates; while gold chloride and
platinic chloride solutions give crystalline precipitates, many of which have
sharp melting points and can be used for the identification of individual
alkaloids. There are a great many specific color reactions for individual
alkaloids, which are important to toxicologists and pharmacists, but which it
would not be desirable to consider in detail here.

The alkaloids are conveniently divided into groups, according to the
characteristic closed-ring arrangements which they contain. The several
closed-ring arrangements which are found in common alkaloids, and upon
which their grouping is based, may be illustrated by the following formulas:

The common alkaloids are distributed in the several groups as follows:

Pyrridine—piperidine group; piperine, coniine, nicotine.
Pyrrolidine group; hygrine and stachydrine.
Tropane group; atropine, hyoscine, cocaine, lupinine.
Quinoline group; quinine, cinchonine, strychnine, brucine.
Isoquinoline group; papaverine, hydrastine, morphine, codeine,
berberine.

The composition and properties of the individual alkaloids have been
extensively studied, because of their medicinal uses. As they have no known
metabolic use to the plants which elaborate them, it will not be worth while
to consider all of these investigations in detail here. The following facts with
reference to certain typical members of each group will serve to illustrate the
general constitution and properties of the alkaloids.

Piperine, C17H19O3, is found in black peppers. Its constitution is
represented by the following formula, the group which is united to the

piperidine ring, in this case, being piperic acid:

Coniine, C8H17N, is found in the umbelliferous plant, Conium
maculatum. Structurally, it is a propyl-piperidine, represented by the
following formula:

Nicotine, C10H14N2, is the alkaloid of tobacco leaves. It is an extremely
poisonous, oily liquid, with a strong odor and a burning taste. Its structural
formula shows it to contain both a pyrridine ring and a pyrrolidine ring,
linked together thus

Hygrine, C7H13NO, from coca leaves, is an acetic acid salt of pyrrolidine,
represented by the following formula:

Atropine and hyoscyamine, C17H23NO3, are optical isomers. Atropine is
an extremely poisonous, white crystalline compound, which is obtained from

deadly nightshade and henbane, and used in medicine, in minute doses, as an
agent for reducing temperature in acute cases of fevers. Structurally, it is a
tropic acid ester of tropane, represented by the following formula:

Cocaine, C17H21NO4, is found in coca leaves. It is a white crystalline
solid, which is largely used as a local anæsthetic for minor surgical
operations. Its structural formula is

It is, therefore, a di-ester of acetic and benzoic acids with tropane.

Cinchonine, C19H22N2O, and quinine, C20H24N2O2, are alkaloids found
in cinchona bark. They are white crystalline solids, which are extensively
used in medicine. They have been shown to contain a quinoline group
combined with modified piperidine groups, as represented in the following
formulas:

Strychnine, C21H22N2O2, brucine, C21H20(OCH3)N2O2, and curarine
are three alkaloids which are present in the seeds of several species of
Strychnos. They are all highly poisonous. Beyond the fact that when they are
hydrolyzed they yield quinoline and indole, their composition is unknown.

Morphine, C17H19NO3, is the chief alkaloid of opium, which is the dried
juice of young pods of the poppy. Both the alcoholic solution of opium
(known as "laudanum") and morphine itself are extensively used in medicine

as narcotics to deaden pain. Morphine has an exceedingly complex structure,
being a combination of an isoquinoline and a phenanthrene nucleus, which is
probably correctly represented by the following formula:

Codeine, C17H18(OCH3)NO2, which is also found in opium, is a methyl
derivative of morphine. Papaverine, laudanosine, narcotine, and narceine
are four other alkaloids found in opium. They each contain an isoquinoline
nucleus, combined by one bond to a benzene ring, with one or more methyl
groups and three or more methoxy (OCH3) groups attached at various points
around the three characteristic rings. The following formula for laudanosine
will illustrate their structure:

The above discussions of the composition of typical alkaloids clearly
indicate the extreme complexity of their molecular structure. It is generally
supposed that they are formed by the decomposition of proteins. But they are
developed in only a few particular species of plants and are always present
in these plants in fairly constant quantities. Hence, it appears that, in these
species, the production of alkaloids is in some way definitely connected with
protein metabolism; but it is certain that this is not a common relationship, as
it is manifested by such a limited number of species of plants, and there is
absolutely no knowledge as to its character and functions. Some authorities
prefer to regard the alkaloids as waste-products of protein metabolism; but
here, again, it is difficult to understand why such products should result in
certain species of plants and not in others.

THE PURINE BASES

This is a group of compounds, widely distributed in both plant and animal
tissues, all of which are derivatives of the compound known as purine,
C5H4N4. All of the naturally occurring compounds of this group may be
regarded as derived from purine, either by the addition of oxygen atoms, or
by the replacing of one or more of its hydrogen atoms with a methyl (CH3)
group or an amino (NH2) group. The following structural formula represents
the arrangement of the purine nucleus, the numbers being used to designate
the nitrogen or carbon atoms to which the additional atoms, or groups, are
attached in the more complex compounds of the group. In purine itself, the
four hydrogen atoms are attached in the 2, 6, 7, and 8 positions.

The double bonds, in each case except those between the 4 and 5 carbon
atoms, are easily broken apart and readjusted, so that other atoms or groups
can be attached to any atom in the nucleus except the 4 and 5 carbon atoms.
In all of the statements with reference to the structure of the purine bases, the
term "oxy" is used to mean an oxygen atom attached by both its bonds to
one of the carbons in the nucleus, instead of its customary use to mean the
monovalent OH group replacing a hydrogen, as in the case of all other
nomenclature of organic compounds. With this understanding, reference to

the numbered nucleus formula above will make plain the structure of all of
the purine bases which are included in the following list:

Hypoxanthine, C5H4N4O, = 6-monoxypurine.
Xanthine, C5H4N4O2, = 2,6-dioxypurine.
Uric acid, C5H4N4O3, = 2,6,8-trioxypurine.
Adenine, C5H3N4NH2, = 6-aminopurine.
Guanine, C5H3N4ONH2, = 2-amino-6-oxypurine.
Theobromine, C5H2N4O2(CH3)2 = 3,7-dimethyl-2,6-dioxypurine, or
dimethyl xanthine.
Theophylline, C5H2N4O2(CH3)2 = 1,3-dimethyl-2,6-dioxypurine.
Caffeine, C5HN4O2(CH3)3 = 1,3,7-trimethyl-2,6-dioxypurine, or
trimethyl xanthine.

In order to make these structural relationships quite clear, the following
formulas for uric acid and for caffeine are presented as typical examples:

Uric acid is found in the excrement of all animals; in the urine of
mammals, and in the solid excrement of birds and reptiles. It is not known to
occur in plants.

Xanthine and hypoxanthine occur in animal urine, and also in the tissues
of both plants and animals.

Adenine and guanine are constituents of all nucleic acids (see below)
and, hence, are found in all plant and animal tissues. Guanine is the chief

constituent of the excrement of spiders, and is found also in Peruvian guano.
It is also a constituent of the scales of fishes.

Caffeine, theophylline, and theobromine are not found in animal tissues,
but are fairly widely distributed in plants. Caffeine and theobromine are the
active constituents of tea leaves and coffee seeds and are found also in cacao
beans and kola nuts. The use of these three compounds in the metabolism of
the plants which elaborate them is wholly unknown. They are not so directly
related to protein metabolism as are the other purine bases.

The purine bases, other than the three mentioned in the preceding
paragraph, are undoubtedly intermediate products in protein metabolism. In
animals, they constitute a large proportion of the waste-products from the
use of proteins in the body. It is not clear that there are similar waste-
products in plant metabolism, however. In both plants and animals, the
purine bases which are a part of the nucleic acids undoubtedly play an
important and essential part in growth, since they form the major proportion
of the nucleus, from which all cell-division proceeds.

THE PYRIMIDINE BASES

These compounds do not occur free in plants; but since they are
constituent groups in the plant nucleic acids (see below), a brief explanation
of their composition is desirable. They are nitrogenous bases, similar to, but
somewhat simpler than, the purine bases. Their general composition and
structural relationships are illustrated by the following typical formulas:

THE NUCLEIC ACIDS

The nuclei of cells are composed almost wholly of complex organic salts,
in which proteins constitute the basic part and nucleic acids the acid part.
These salts, or esters, are known under the general name "nucleoproteins."
The composition of the proteins is discussed in detail in the following
chapter, and it seems desirable to present a brief discussion of the

constitution of the nucleic acids here; although they are essentially acids
rather than vegetable bases.

The nucleic acids are complex compounds consisting of a carbohydrate,
phosphoric acid, two purine bases, and two pyrimidine bases. So far as is
known, all animal nucleic acids are identical and all plant nucleic acids are
identical; but those of plant origin differ from those found in animal cells in
the character of the carbohydrate and that of one of the pyrimidine bases
which are present in the molecule, as shown in the following tabulation of
their composition:

Animal nucleic acid Plant nucleic acid
Phosphoric acid Phosphoric acid
Hexose (levulose) Pentose (d-ribose)
Guanine Guanine
Adenine Adenine
Cytosine Cytosine
Thymine Uracil

The structure of the plant nucleic acid may be represented by the
following formula:

That this is probably a correct representation of the general arrangement
in this compound, is indicated by the fact that by different methods of
hydrolysis it is possible to split off either the purine and pyrimidine bases,
leaving a carbohydrate ester of phosphoric acid; or the phosphoric acid,
leaving carbohydrate combinations with the nitrogenous bases.

Nucleic acid, prepared from animal glands which contain large
proportions of it, is a white powder, which is insoluble in water, but when
moistened forms a slimy mass. It is almost insoluble in alcohol, but

dissolves readily in alkaline solutions, forming a colloidal solution which
readily gelatinizes (see Chapter on Colloids). Solutions of nucleic acids are
optically active, probably because of the carbohydrate constituents.

From their structure and properties, it is apparent that nucleic acids are on
the border line between carbohydrates, plant amines, and proteins. They
undoubtedly play an important part, both in cell-growth and in the synthesis
of proteins from carbohydrates and ammonium compounds.

References

Barger, Geo.—"The Simpler Natural Bases," 215 pages,
Monographs on Biochemistry, London, 1914.

Fischer, E.—"Untersuchungen in der Puringruppe, 1882-1906,"
608 pages, Berlin, 1907.

Henry, T. A.—"The Plant Alkaloids," 466 pages, Philadelphia,
1913.

Jones, W.—"The Nucleic Acids," 118 pages, Monographs on
Biochemistry, London, 1914.

Pictet, A.—"La Constitution Chimique des Alcaloides
Vegetaux," 421 pages, Paris, 1897 (2d ed.).

Vaughan, V. C. and Novy, F. G.—"Ptomaines, Leucomaines,
Toxins and Antitoxins," 604 pages, Philadelphia, 1896, (3d ed.).

Winterstein, E. and Trier, G.—"Die Alkaloide," 340 pages,
Berlin, 1910.

CHAPTER XIII

PROTEINS

The proteins are the most important group of organic components of
plants. They constitute the active material of protoplasm, in which all of the
chemical changes which go to make up the vital phenomena take place.
Combined with the nucleic acids, they comprise the nucleus of the cell,
which is the seat of the power of cell-division and, hence, of the growth of
the organism. Germ-cells are composed almost exclusively of protein
material. Hence, it is not an over-statement to say that proteins furnish the
material in which the vital powers of growth and repair and of reproduction
are located. A recognition of their importance is reflected in the use of the
name "protein," which comes from a Greek word meaning "pre-eminence,"
or "of first importance."

In addition to the proteins which constitute the active protoplasm, plants
also contain large amounts of reserve, or stored, proteins, especially in the
seeds. In the early stages of growth, the proteins are present in largest
proportions in the vegetative portions of the plant; but as maturity
approaches, a considerable proportion of the protein material is transferred
to the seeds.

GENERAL COMPOSITION OF PROTEINS

The plant proteins are fairly uniform in their percentage composition. The
analyses of some sixteen different plant proteins show the following
maximum limits of percentages of the different chemical elements which
they contain: Carbon, 50.72-54.29; hydrogen, 6.80-7.03; nitrogen, 15.84-
19.03; oxygen, 20.86-24.29; sulfur, 0.17-1.09. Animal proteins vary more
widely, both in percentage composition and in properties, than do those of
plant origin.

Protein molecules are very large and, in the case of the so-called
"conjugated proteins" in particular, their structure is very complex. The
molecular weight of some of the proteins has been determined directly, in
the case of those particular ones which can be prepared in proper form for
the usual determination of molecular weight by the osmotic pressure
method; and has been computed for various others, from the percentage of
sulfur found on analysis, or (in the case of the hæmoglobin of the blood)
from the proportion by weight of oxygen absorbed. From these
determinations and computations, the following formulas for certain typical
proteins have been calculated: for zein (from Indian corn),
C736H1161N184O208S3; for gliadin (from wheat), C685H1068N196O211S5; for
casein (from milk), C708H1130N180O224S4P4; for egg-albumin,
C696H1125N175O220S8. These few examples will serve to illustrate the
enormous size and complexity of the protein molecule. The conjugated
proteins are still more complex than the simple proteins whose formulas are
here presented.

Fortunately for the purposes of the study of the chemistry of the proteins,
however, it has been found that most of the common plant proteins, known
as the "simple proteins," can easily be hydrolyzed into their constituent unit
groups, which are the comparatively simple amino-acids, whose composition

and properties are well understood. A study of the results of the hydrolysis of
some twenty common plant proteins has shown that it is rarely possible to
recover the amino-acids in sufficient quantities to account for a full 100 per
cent of the material used, the actual percentage of amino-acids recovered
usually totaling from 60 to 80 per cent. The remaining material is supposed
to be also composed of amino-acids which are linked together in some
arrangement which is not broken apart by any method of hydrolysis which
has yet been devised. This view is borne out by the fact that substances
which exhibit all the characteristic properties of proteins have been
artificially synthetized, by using only amino-acid compounds. Animal
proteins often show a much larger proportion of unhydrolyzable material
than do plant proteins.

AMINO-ACIDS AND PEPTID UNITS

The products of hydrolysis of the common simple proteins are all amino-
acids. These are ordinary organic acids with one (or more) of the hydrogen
atoms of the alkyl group replaced by a —NH2 (or sometimes by a —NH—)
group. They may be regarded as ammonia, NH3, with one of its hydrogen
atoms replaced by an acid radical; or as the acid with one of its hydrogens
replaced by the NH2 group. For example, an amino-acid derived from acetic
acid, CH3·COOH, is glycine, or amino-acetic acid, CH2NH2·COOH; from
propionic acid, CH3·CH2·COOH, there may be obtained either α-amino-
propionic acid, CH3·CHNH2·COOH, or β-amino-propionic acid,
CH2NH2·CH2·COOH, etc.

All of the amino-acids which result from the hydrolysis of proteins are α-
amino-acids, that is to say, the NH2 group is attached to the α-carbon atom,

i.e., the one nearest to the COOH group. Hence, the general formula for all
the amino-acids which are found in plants is R·CHNH2·COOH.

These amino-acids contain both the basic NH2 group and the acid COOH
group. For this reason, they very easily unite together, in the same way that
all acids and bases unite, to form larger molecules, the linkage taking place
between the basic NH2 group of one molecule and the acid COOH group of
the other, as indicated by the following equation:

It is obvious that the compound thus formed still contains a free NH2
group and a free COOH group, and is, therefore, capable of linking to
another amino-acid molecule in exactly the same way; and so on
indefinitely. In actual laboratory experiments, as many as eighteen of these
amino-acid units have been caused to unite together in this way, and the
resulting compounds thus artificially prepared have been found to possess
the characteristic properties of natural proteins.

These artificially prepared, protein-like, substances have been called
"polypeptides," and the individual amino-acids which unite together to form
them are called "peptides." Thus, a compound which contains three such
units linked together is called a "tripeptid"; one which contains four, a
"tetrapeptid." The use of the term "peptid" was suggested by the fact that

these amino-acids are produced from the hydrolysis of proteins by the
digestive enzyme pepsin.

The peptid units of any such complex as those which have been referred
to in the preceding paragraphs may be linked together in a great variety of
ways. Thus, in a tetrapeptid containing units which may be designated by the
letters a, b, c, and d, the arrangement may be in the orders abcd, bacd, acbd,
dbca, etc., etc. Similarly, the same peptid unit may appear in the molecule in
two or more different places. Hence, the number of possible combinations of
amino-acids into protein molecules is very great. Further, it is possible that
the peptid units in natural proteins may be united together through other
linkages than the one illustrated above, as they often contain alcoholic OH
groups in addition to the basic NH2 groups, and these OH groups may form
ester-linkages with the acid (COOH) groups of other units. Still other acid
and basic groups are present in some of the amino-acids which have been
found in natural proteins, so that the possibility of variation in the polypeptid
linkages is almost limitless.

INDIVIDUAL AMINO-ACIDS FROM PROTEINS

About twenty different amino-acids have been isolated from the products
of hydrolysis of natural proteins, and this number is being added to from
time to time, as the methods of isolation and identification of these
compounds are improved. Many of these same amino-acids have been found
in free form in plant tissues, particularly in rapidly growing buds, or shoots,
or in germinating seeds, where they undoubtedly exist as intermediate
products in the transformation of proteins into other types of compounds.

These amino-acids, grouped according to the characteristic groups which
they contain, are as follows:

A. Monoamino-monocarboxylic acids:
Glycine, C2H5NO2, CH2NH2·COOH, amino-acetic acid.
Alanine, C3H7NO2, CH3·CHNH2·COOH, amino-propionic acid.
Serine, C3H7NO3, CH2OH·CHNH2·COOH, oxy-amino-propionic
acid.

Valine, C5H11NO2, , amino-
isovalerianic acid.

Leucine, C6H13NO2, , amino-
isocaproic acid.

Isoleucine, C6H13NO2, , amino-
methylethyl-propionic acid.

Phenylalanine, C9H11NO2, , phenyl-amino-
propionic acid.

Tyrosine, C9H11NO3, , paraoxy-
phenylalanine.
Cystine, C6H12N2O4S2, HOOC·CHNH2·CH2S—
SH2C·CHNH2·COOH, di(thio-amino-propionic acid).
B. Monoamino-dicarboxylic acids:
Aspartic acid, C4H7NO4, HOOC·CH2·CHNH2·COOH, mino-
succinic acid.
Glutamic acid, C5H9NO4, HOOC·CH2·CH2·CHNH2·COOH,
amino-glutaric acid.

C. Diamino-monocarboxylic acids:
Ornithine, C5H12N2O2, H2N·CH2·CH2·CH2·CHNH2·COOH, di-
amino-valerianic acid.

Lysine, C6H14N2O2, H2N·CH2·CH2·CH2·CH2·CHNH2·COOH,
di-amino-caproic acid.
Arginine, C6H14N4O2,

, guanidine-
amino-valerianic acid.
Di-amino-oxysebacic acid, C11H12N2O3.
Di-amino-trioxydodecanic acid, C12H26N2O3.
D. Monoimido-monocarboxylic acids:

Proline, C5H9NO2, , pyrrolidine-carboxylic
acid.
Oxyproline, C5H9NO3, proline with one (OH) group.
E. Monoimido-monoamino-monocarboxylic acids:

Histidine, C6H9N3O2, , imidazole-amino-
propionic acid.

Tryptophane, C11H12N2O2, , indole-
amino-propionic acid.

As has been said, other amino-acids are being found, from time to time, as
additional proteins are examined, or as better methods of examination of the
cleavage products of the natural proteins are devised.

COMPOSITION OF PLANT PROTEINS

The distribution of the different amino-acids in some of the different plant
proteins which have been examined in this way is shown in the following
table:

Globulin
Gliadin Hordein Zein Legumin Edestin Amandin
(squash
(whaet). (barley). (corn). (vetch). (hemp). (almonds).
seed).
Glycine 0.02 0.00 0.00 0.39 3.80 0.57 0.51
Alanine 2.00 0.43 9.79 1.15 3.60 1.92 1.40
Valine 0.21 0.13 1.88 1.36 6.20 0.26 0.16
Leicine 5.61 5.67 19.55 8.80 14.50 7.32 4.45
Proline 7.06 13.73 9.04 4.04 4.10 2.82 2.44
Phenylalanine 2.35 5.03 6.55 2.87 3.09 3.32 2.53
Aspartic acid 0.58 ..... 1.71 3.21 4.50 3.30 5.42
Glutamic acid 42.98 43.19 26.17 18.30 18.84 12.35 23.14
Serine 0.13 ? 1.02 ? 0.33 ? ?
Cystine 0.45 ? ? ? 1.00 0.23 ?
Tyrosine 1.20 1.67 3.55 2.42 2.13 3.07 1.12
Arginine 3.16 2.16 1.55 11.06 14.17 14.44 11.85
Histidine 0.61 1.28 0.43 2.94 2.19 2.63 1.58
Lysine ..... ..... ..... 3.99 1.65 1.99 0.70
Tryptophane present present present present present present present
Ammonia 5.11 4.87 3.64 2.12 2.28 1.55 3.70
71.46 78.16 85.27 62.65 82.38 55.77 59.00

At the time when these analyses were made, a method for the quantitative
estimation of tryptophane had not been devised, although one is now
available. The addition of the percentages of tryptophane and of other amino-
acids for which methods of determination are not yet known, would bring the
total, in each case, more nearly up to the full 100 per cent. These data will
serve to show how widely the different plant proteins vary in the proportions
of the different amino-acids which they contain. Animal proteins have been
found to be still more variable in composition.

In the use of the proteins as food for animals, it appears that the different
amino-acids are in some way connected with the different physiological
functions which the proteins have to perform in the animal body: thus,
tryptophane is absolutely essential to the maintenance of life, but does not

promote growth; lysine, on the other hand, definitely promotes growth, so that
animals which have been maintained without any increase in weight for many
months immediately begin to grow when furnished with a diet in which lysine
is a constituent; while arginine seems to be definitely associated with the
reproductive function; and cystine, with the growth of hair, feathers, etc. It is
not known whether there is any similar relation of amino-acids to the
functions of different proteins in plant metabolism.

The separation of the individual amino-acids from the mixture which results
from the hydrolysis of any given protein is a long and tedious process and, at
best, yields only moderately satisfactory results. For that reason, it has
recently been almost entirely abandoned in favor of the separation devised by
Van Slyke, which divides the total nitrogenous matter in the mixture resulting
from the hydrolysis of a protein into the following groups; ammonia N, humin
(or melanin) N, cystine N, arginine N, histidine N, lysine N, amino N of the
filtrate, and non-amino N of the filtrate. These groups can be conveniently and
fairly accurately separated out of the hydrolysis mixture, by means of various
precipitating agents, and the quantity of N in the several precipitates
determined by the usual Kjeldahl method. The actual process for these
separations need not be discussed here, as it is given in detail in all standard
text-books dealing with the methods of biochemical analysis. The distribution
of the nitrogen in any given protein into these various groups is characteristic
for that particular protein, and the process serves both as a means of
identification of individual proteins and a method for tracing their changes
through various vital, or biochemical, transformations.

GENERAL PROPERTIES OF THE PROTEINS

Individual proteins differ slightly in their characteristics, but in general they
are all alike in the following physical and chemical properties.[5]

Physical Properties.—(1) The proteins are all colloidal in character, that is,
they form solutions in water, out of which they cannot be dialyzed through
parchment, or other similar membranes. (2) All natural proteins, when in
colloidal solution, may be coagulated, forming a semi-solid gel, which cannot
again be rendered soluble except by decomposition. The most familiar
example of this type of coagulation is that of egg-albumin, when eggs are
cooked. This coagulation may be produced by heat, by the action of certain
enzymes, or by the addition of alcohol to the solution. (3) All solutions of
plant proteins are optically active, rotating the plane of polarized light to the
left, in every case. (4) Proteins are precipitated out of their solutions, without
change in the composition of the protein, by saturating the solution with
various neutral salts of the alkali, or alkaline earth, metals, such as sodium
chloride, ammonium sulfate, magnesium sulfate, etc. This is only another way
of saying that the proteins are insoluble in strong salt solutions. Separation
from solution by the addition of salts is different from coagulation by heat,
etc., as in this case simple dilution of the salt solution will cause the protein to
redissolve, whereas a coagulated protein cannot be redissolved without some
change in its composition.

Chemical Properties. (1) Precipitation reactions.—The proteins have both
acid and basic properties (due to the presence in their molecules of both free
NH2 groups and free COOH groups). Bodies of this kind are known as
"amphoteric electrolytes," since they yield both positive and negative ions, if
dissociated. The proteins readily form salts, which are generally insoluble in
water, with strong acids. For this reason, they are generally precipitated out of
solution by the addition of the common mineral acids. They are also
precipitated by many of the "alkaloidal reagents," to which reference has been
made in the preceding chapter, namely, phosphotungstic, phosphomolybdic,
tannic, picric, ferrocyanic, and trichloracetic acids, the double iodide of
potassium, mercuric iodide, etc. The precipitates produced by strong mineral
acids are often soluble in excess of the acid, with the formation of certain so-

called "derived proteins," which are probably products of the partial
hydrolysis of the protein.

The proteins are also precipitated out of solution by the addition of small
amounts of salts of various heavy metals, such as the chlorides, sulfates, and
acetates of iron, copper, mercury, lead, etc. This precipitation is different than
that caused by the saturation of the solution with the salts of the alkali metals,
as in this case the metal unites with the protein to form definite, insoluble
salts, which cannot be redissolved except by treatment with some reagent
which removes the metal from its combination with the protein (hydrogen
sulfide is commonly used for this purpose).

(2) Color reactions.—Certain specific groups which are present in most
proteins give definite color reactions with various reagents. It is apparent that
any individual protein will respond to a particular color reaction, or will not do
so, depending upon whether the particular group which is responsible for the
color in question is present in that particular protein. Color reactions to which
most of the common plant proteins respond are the following ones:

(a) Biuret Reaction.—Solutions of copper sulfate, added to an alkaline
solution of a protein, give a bluish-violet color if the substance contains two,
or more, —CONH— groups united together through carbon, nitrogen, or
sulfur atoms. Inasmuch as most natural proteins contain several such groups,
the biuret reaction is a very general test for proteins.

(b) Millon's Reaction.—A solution of mercuric nitrate containing some free
nitrous acid (Millon's reagent) produces a precipitate which turns pink or red,
whenever it is added to a solution which contains tyrosine, or a tyrosine-
containing protein.

(c) Xanthoproteic Acid Reaction.—This is the familiar yellow coloration
which is produced whenever nitric acid comes in contact with animal flesh. It

is caused by the action of nitric acid on tyrosine. The color is intensified by
heating, and is changed to orange-red by the addition of ammonia.

(d) Adamkiewicz's Reaction.—If concentrated sulfuric acid be added to a
solution of a protein to which some acetic acid (or better, glyoxylic acid) has
previously been added, a violet color is produced. This color will appear as a
ring at the juncture of the two liquids, if the sulfuric acid is poured carefully
down the sides of the tube, or throughout the mixture if it is shaken up. It
depends upon the interaction of the glyoxylic acid (which is generally present
as an impurity in acetic acid) upon the tryptophane group, and is therefore
given by all proteins which contain tryptophane.

(e) Molisch's reaction for furfural will be shown by those proteins which
contain a carbohydrate group. In applying this test, the solution to be tested is
first treated with a few drops of an alcoholic solution of α-naphthol, and then
concentrated sulfuric acid is poured carefully down the sides of the test-tube.
If carbohydrates are present, either free or as a part of a protein molecule, a
red-violet ring forms at the juncture of the two liquids.

(f) Sulfur Test.—If a drop of a solution of lead acetate be added to a solution
containing a protein, followed by sufficient sodium hydroxide solution to
dissolve the precipitate which forms, and the mixture is heated to boiling, a
black or brown coloration will be produced if the protein contains cystine, the
sulfur-containing amino-acid.

FOOTNOTES:

[5 Since the proteins are essentially colloidal in nature, many of the terms used in the
] discussions of their properties, and these properties themselves, will be better understood after
the chapter dealing with the colloidal condition of matter has been studied. A more logical
arrangement so far as the systematic study of these properties is concerned would be to take up

chapter XV before undertaking the study of the proteins (this order is actually followed in some
texts on Physiological Chemistry). But from the standpoint of the consideration of the various
groups of organic components of plants, it seems a better arrangement to consider these groups in
sequence, and then to discuss the various physical-chemical phenomena which govern their
activity. However, it is recommended that the student refer at once to Chapter XV for an
explanation of any terms used here, which may not be familiar to him; and that after the study of
Chapter XV, he return to this chapter dealing with the proteins for an illustrative study of the
applications of the principles presented there.

THE CLASSIFICATION OF THE PROTEINS

Formerly, the classification of proteins was based almost wholly upon their
solubility and coagulation reactions. More recently, since their products of
hydrolysis have been extensively studied, their classification has been
modified, in attempts to make it correspond as closely as possible to their
chemical constitution and physical properties. As knowledge of these matters
progresses, the schemes of classification change. On that account, no one
definite scheme is universally used. For example, the English system varies
considerably from the one commonly used by American biochemists, which is
the one presented below.

The proteins are divided into three main classes, as follows:

1. Simple proteins, which yield only amino-acids when hydrolyzed.
2. Conjugated proteins, compounds of proteins with some other non-protein
group.
3. Derived proteins, decomposition products of simple proteins.

The first two of these classes comprise all the natural proteins; while the
third includes the artificial polypeptides and proteins which have been
modified by reagents.

These major classes are further subdivided into the following sub-classes,
which depend in part upon the solubilities of the individual proteins, and in
part upon the nature of their products of hydrolysis:

1. The Simple Proteins
A. Albumins—soluble in water and dilute salt solutions, coagulated by
heat.
B. Globulins—insoluble in water, soluble in dilute salt solutions,
coagulated by heat.
C. Glutelins—insoluble in water or dilute salt solutions, soluble in
dilute acids or alkalies, coagulated by heat.
D. Prolamins—insoluble in water, etc., soluble in 80 per cent alcohol.
E. Histones—soluble in water, insoluble in ammonia, not coagulated
by heat.
F. Protamines—soluble in water and ammonia, not coagulated by heat,
yielding large proportions of diamino-acids on hydrolysis.
G. Albuminoids—insoluble in water, salt solutions, acids, or alkalies.

2. Conjugated Proteins
A. Chromoproteins—compounds of proteins with pigments.
B. Glucoproteins—compounds of proteins with carbohydrates.
C. Phosphoproteins—proteins of the cytoplasm, containing phosphoric
acid.
D. Nucleoproteins—proteins of the nucleus, containing nucleic acids.
E. Lecithoproteins—compounds of proteins with phospholipins.
F. Lipoproteins—compounds of proteins with fats, existence in nature
doubtful, artificial forms easily prepared.

3. Derived Proteins
A. Primary protein derivatives.
a. Proteans—first products of hydrolysis, insoluble in water.
b. Metaproteins—result from further action of acids or alkalies,
soluble in weak acids and alkalies, but insoluble in dilute salt
solutions.
c. Coagulated proteins—insoluble forms produced by the action
of heat or alcohol.
B. Secondary protein derivatives.

a. Proteoses—products of hydrolysis, soluble in water, not
coagulated by heat, precipitated by saturation of solution with
ammonium sulfate.
b. Peptones—products of further hydrolysis soluble in water, not
coagulated by heat, not precipitated by ammonium sulfate, give
biuret reaction.
c. Peptides—individual amino-acids, or poly-peptides, may or
may not give biuret reaction.

The plant proteins which have been investigated, thus far, fall into these
groups as follows:

1A. Albumins
found in seeds
Leucosin, wheat, rye and barley.
the of
pea, horse-bean, vetch, soy-bean, lentil,
Legumelin, " "
cowpea, adzuki-bean.
Phaselin, " " kidney-bean.
Ricin, " " castor-bean.

1B. Globulins
found in seeds
Legumin, pea, horse-bean, lentil and vetch.
the of
Vignin, " " cowpea.
Glycinin, " " soy-bean.
Phaseolin, " " beans (Phaseolus spp.)
Conglutin, " " lupines.
Vicilin, " " pea, horse-bean, lentil.
Corylin, " " hazel nut.
Amandin, " nuts of almond and peach.
seeds
Juglansin, " walnut and butternut.
of
Excelsin, " " Brazil nut
Edestin, " hemp seed.
Avenalin, " oats.
Maysin, " corn.

Castanin, " seeds of European chestnut.
And, crystalline globulins found in the seeds of flax, squash, castor-bean,
sesame, cotton,
sunflower, radish, rape, mustard, and in cocoanuts, candlenuts, and
peanuts.

1C. Glutelins
found in seeds
Glutenin, wheat.
the of
Oryzenin, " " rice.

1D. Prolamins
found in seeds
Gliadin, rye, wheat, with glutenin forms "gluten."
the of
Hordein, " " barley.
Zein, " " corn.

1E-1G. Histones, Protamines and Albuminoids.—So far as is now known,
no representatives of these classes are found in plants.

2. Conjugated Proteins.—There is no conclusive evidence of the existence
in plants of any of the conjugated proteins, other than the nucleoproteins and
the chromoproteins, the composition and properties of which have been
discussed in previous chapters. The nucleoproteins undoubtedly occur in the
embryos of many, if not all, seeds.

3. Derived Proteins.—Representatives of the various types of derived
proteins are undoubtedly found as temporary intermediate products in plants,
both as products of hydrolysis produced during the germination of seeds and
as intermediate forms in the synthesis of proteins. So far as is known,
however, they do not occur as permanent forms in any plant tissues. They have
been prepared in large numbers and quantities, by the hydrolysis of the natural
proteins and the artificial synthesis of polypeptides.

In the present state of our knowledge concerning the functioning of the
proteins, no significance in the physiology of plant life, or metabolism, is to be
attached to the particular type of protein material which it contains, at least so
far as the simple proteins of the cytoplasm are concerned.

DIFFERENCES BETWEEN PLANT AND ANIMAL
PROTEINS

A much larger variety of protein materials is found in animal tissues than in
plants. This is undoubtedly because different animal organs perform so much
more varied physiological functions than do those of plants. Three groups of
simple proteins, the histones, the protamines, and the albuminoids, which are
quite common in animal tissues, are entirely unknown in plants. Further,
conjugated proteins of greater complexity and more varied structure are found
in animal tissues, especially in the brain, nerve-cells, etc., than in plants.

Plant proteins, in general, usually contain larger proportions of proline and
of glutamic acid than are found in animal proteins; also more arginine than is
found in any of the animal proteins except the protamines, which contain as
high as 85 per cent of this amino-acid.

Of the twenty-five plant proteins which have thus far been hydrolyzed and
studied from this standpoint, all contained leucine, proline, phenylalanine,
aspartic acid, glutamic acid, tyrosine, histidine, and arginine; two gave no
glycine; two others, no alanine; four contained no lysine; and one, no
tryptophane. Zein, the principal protein of corn contains no glycine, lysine, or
tryptophane. It is not sufficient to support animal life and promote growth, if
used as an exclusive source for protein for food.

THE EXTRACTION OF PROTEINS FROM PLANT
TISSUES

Since proteins are indiffusible, it is essential that the cell-walls of the tissue
shall be thoroughly ruptured as the first step in any process for the extraction
of these compounds from plant tissues. This is usually accomplished by
grinding the material as finely as possible, preferably with the addition of
sharp quartz sand, or broken glass, to aid in the tearing of the cell-wall
material.

The solvent to be used in extracting the proteins from this finely ground
material depends upon the nature and solubility of the proteins which are
present, and also upon whether it is desired to separate the proteins which may
be present in the plant, during the process of the extraction. A glance at the
scheme of classification of the proteins will show the following solubilities
which serve as a guide to the procedure to be followed: (a) proteoses,
albumins, and some globulins may be extracted with water; (b) globulins and
most of the water-soluble proteins may be extracted by using a 10 per cent
solution of common salt; (c) prolamines are extracted by 70-90 per cent
alcohol; glutelins and prolamins dissolve in dilute acids or dilute alkali.

A common procedure is to extract groups (a) and (b), using a 10 per cent
salt solution as the solvent, and then to separate the albumins, globulins, etc.,
from this solution by suitable precipitants; then to treat the material with 80
per cent alcohol, to extract the prolamines; and finally with dilute alkali, to
extract the glutelins. The dissolved proteins in each extract can be
subsequently purified by dialysis, precipitation, etc. The insoluble proteins can
be studied only after removing the other materials associated with them in the
tissue, by suitable mechanical or chemical means.

THE SYNTHESIS OF PROTEINS IN PLANTS

The synthesis of proteins in plants is not a process of photosynthesis, as it
can take place in the dark and in the absence of chlorophyll, or any other
energy-absorbing pigment. However, protein-formation normally takes place
in conjunction with carbohydrate-formation. The carbon, hydrogen, and
oxygen necessary for protein synthesis are undoubtedly obtained from
carbohydrates. The nitrogen and sulfur come from the salts absorbed from the
soil through the roots and brought to the active cells in the sap. Atmospheric
nitrogen cannot be used by plants for this purpose, except in the case of certain
bacteria and other low plants, notably the bacteria which live in symbiosis
with the legumes in the nodules on the roots of the host plants. In general, the
sulfur must come in the form of sulfates and the nitrogen in the form of
nitrates; although many plants can make use of ammonia for protein-
formation. Presumably, the nitrate nitrogen must be reduced in the plant to
nitrites, and then to ammonia form, in order to enter the amino-arrangement
required for the greater proportion of the protein nitrogen.

The mechanism by which ammonia nitrogen becomes amino-acids in the
plant is not understood. Artificial syntheses of amino-acids, by the action of
ammonia upon glyoxylic acid and sorbic acid, both of which occur in plants
and may be obtained by the oxidation of simple sugars, have been
accomplished, and it seems probable that similar reactions in the plant
protoplasm may give rise to the various amino-acids which unite together to
form proteins. Nothing is known, however, of the process by which the more
complicated closed-ring amino-acid compounds, such as proline, histidine, or
tryptophane, are synthetized.

The condensation of amino-acids into proteins, or the reverse
decomposition, is very readily accomplished in all living protoplasm, under
the influence of special protein-attacking enzymes, which are almost
universally present in the cytoplasm. These reactions in connection with the

proteins are similar to the easy transformation of sugars to starches, and vice
versa, under the action of the corresponding carbohydrate-attacking enzymes.

PHYSIOLOGICAL USES OF PROTEINS

There can be no doubt that the all-important rôle of proteins, in either plant
or animal tissue, is to furnish the colloidal protoplasmic material in which the
vital phenomena take place. Their occurrence in seeds, and other storage
organs, is, of course, in order to provide the protoplasm-forming material for
the young seedling plant.

They are, moreover, the source for the material which goes into some of the
secretion groups of organic compounds; as they are easily broken down by
various agents of decomposition into nitrogen-free alcohols, aldehydes, and
acids, which produce the essential oils, pigments, etc.

Much, if not all, of their physiological activity is due to their colloidal
nature, the importance and effects of which will be more apparent after the
chapters dealing with the colloidal condition of matter and with the physical
chemistry of protoplasm have been studied.

References

Abderhalden, E.—"Neuere Ergebnisse auf dem Gebiete der
Speziellen Eiweisschemie," 128 pages, Jena, 1909.

Fischer, E.—"Untersuchungen über Aminosäuren, Polypeptide,
und Proteine, 1899-1906," 770 pages, Berlin, 1906.

Mann, G.—"Chemistry of the Proteids," 606 pages, London, 1906.

Osborne, T. B.—"The Vegetable Proteins," 138 pages,
Monographs on Biochemistry, London, 1909.

Plimmer, R. H. A.—"The Chemical Constitution of the Proteins,
Part I, Analysis," 188 pages; and "Part II, Synthesis, etc." 107
pages, Monographs on Biochemistry, London, 1917. (3d ed.).

Robertson, T. B.—"The Physical Chemistry of the Proteins," 477
pages, New York, 1918.

Schryber, S. B.—"The General Characters of the Proteins," 86
pages, Monographs on Biochemistry, London, 1909.

Underhill, F. P.—"The Physiology of the Amino-acids," 169
pages, 13 figs. 1 plate. Yale University Press, 1915.

CHAPTER XIV

ENZYMES AND THEIR ACTION

The characteristic difference between the reactions of inorganic compounds
and those of organic substances lies in the rapidity, or velocity, of the chemical
changes involved. Speaking generally chemical reactions take place between
substances which are in solution, so that they may come into sufficiently
intimate contact that chemical action between them can take place. There are,
of course, occasional examples of reactions between dry solids, such as the
explosion of gunpowder, etc., but the general rule is that reacting materials
must be in either colloidal or true solutions.

Inorganic materials, when dissolved in water, usually ionize very readily.
That is, they are not only disintegrated into individual molecules, but a
considerable proportion of these molecules separate into their constituent ions.
When solutions containing ionized compounds are brought together,
conditions for chemical interaction are ideal, and the reaction proceeds with
such tremendous rapidity as to be completed almost instantaneously, in most
cases.

Organic compounds, on the other hand, ionize only very slowly, if at all.
Hence, reactions between organic compounds, even when they are in solution,
proceed very slowly unless carried on at high temperatures, under increased

pressure, or under the influence of some catalytic agent. Even under the
stimulation of these reaction-accelerating agencies, most chemical changes in
organic compounds when carried on in the laboratory, require several hours or
even days and sometimes weeks, for their completion. But when similar
reactions take place in living organisms, they proceed with velocities which
resemble those of inorganic compounds in the laboratory. This difference
between the velocity of organic reactions when carried on under artificial
conditions in the laboratory (often spoken of as "in vitro") as compared with
that of the same reactions when they take place in a living organism ("in
vivo"), is due to the universal presence in the living protoplasm of certain
organic catalysts, known as enzymes.

ENZYMES AS CATALYSTS

The phenomenon known as "catalysis" is of common occurrence in both
inorganic and organic chemistry. The effect of a small amount of manganese
dioxide in aiding in the liberation of oxygen from potassium chlorate is an
example which is familiar to all students of elementary chemistry. Similarly,
spongy platinum accelerates the oxidation of sulfur dioxide to sulfur trioxide,
in the commercial manufacture of sulfuric acid. Again, the hydrolysis of
sucrose into fructose and glucose proceeds very slowly in the presence of
water alone, but if a little hydrochloric acid or sulfuric acid be added to the
solution, the velocity of the hydrolysis is enormously accelerated. Many other
examples of the accelerating effect of various chemicals upon reactions into
which they do not themselves enter, might be cited.

The essential features of all such catalytic actions are: (1) the velocity of the
reaction is greatly altered, usually accelerated; (2) the catalytic agent does not
appear as one of the initial substances, or end-products, of the reaction, and is
not itself altered by the chemical change which is taking place; (3) the

accelerating effect is directly proportional to the amount of the catalyst which
is present; (4) relatively small amounts of the catalyst produce very large
results in the reacting mixture; and (5) the catalysts cannot themselves initiate
reactions, but only influence the velocity of reactions which would otherwise
take place at a different rate (usually much more slowly) in the absence of any
catalytic agent.

Enzymes conform to all of these properties of catalysts, and are commonly
defined as the "catalysts of living matter." They are almost universally present
in living organs of every kind, and perform exceedingly important functions,
both in the building-up of synthetic materials and in the rendering soluble of
the food of both plants and animals, so that it can be translocated from place to
place through the tissues of the organism.

Enzymes differ from inorganic catalysts in being destroyed by heat, in not
always carrying the reaction to the same stage as does the inorganic catalyst
which may accelerate the same reaction, and in producing different changes in
the same substance by different enzymes.

The name "enzyme" comes from Greek words meaning "in yeast," as the
nature and effect of the enzyme involved in the alcoholic fermentation of
sugars by yeast were those which were first recognized and understood. It was
at first thought, by Pasteur and his students, that fermentation is the direct
result of the life activities of the yeast plant. Later, it was found that water
extracts from sprouted barley, from almond seeds, and from the stomach,
pancreas, etc., were able to bring about the decomposition of starch, of
amygdalin, and of proteins, respectively, in a way which seemed to be quite
comparable to the fermentative action of yeasts. Hence, it was thought that
there were two varieties of active agents of this kind, one composed of living
cells and the other non-living chemical compounds, and these were called the
"organized ferments" and the "unorganized ferments," respectively. However,
in 1897, Büchner found that by grinding yeast cells with sharp sand until they

were completely disintegrated and then submitting the mass to hydraulic
pressure, he could obtain a clear liquid, entirely free from living cells, which
was just as active in producing fermentation as was the yeast itself. This
discovery paved the way for a long series of investigations, which have
conclusively demonstrated that there is no distinction between "organized"
and "unorganized" ferments, that all living organisms perform their
characteristic functions by means of the enzymes which they contain, and that
these enzymes can bring about their characteristic catalytic effects outside the
cell, or tissue which elaborates them, just as well as within it, provided only
that the conditions of temperature, acidity or alkalinity of the medium, etc., are
suitable for the particular enzyme action which is under consideration.

GENERAL PROPERTIES OF ENZYMES

Since enzymes are catalysts, it is plain that an accurate description of their
activity should, in each case, refer to the influence which they exert upon
some definite reaction velocity. But since the phrases necessary to describe
such an effect are cumbersome and inconvenient, and since most of the
reactions which are accelerated by the catalytic action of enzymes are either
simple hydrolyses, changes in oxygen content, or other simple decompositions
or condensations, which will otherwise proceed so slowly as to be practically
negligible, it is customary to speak of the enzyme as "acting upon" the
material in question. It should be understood, however, that this is a
misstatement, as the enzyme cannot actually initiate a reaction, or "act upon"
any substance; it only acts as a catalyzer to accelerate the action of water,
oxygen, etc., upon the material in question.

Generally speaking, most enzymes are colloidal in form and, hence, do not
diffuse through membranes such as the cell-walls. Some of them perform their
characteristic functions only within the cell, or organ, which elaborates them,

and can be obtained outside these tissues for purposes of study only by first
rupturing the cell-wall or other membrane with which they are surrounded.
Such enzymes are known as "intracellular." Others are regularly secreted by
glands which discharge them onto other organs, as the stomach or intestines of
animals, where they perform their useful functions; or, as in the case of
germinating seeds, they move to other parts of the organ, and can be extracted
from the tissue by simple treatment with water. These are known as the
"extracellular" enzymes.

Enzymes are specific in their action. Any given enzyme affects only a single
reaction; or at most acts only upon a single group of compounds which have
similar molecular configuration. Usually it is only a single compound whose
decomposition is accelerated by the action of a particular enzyme; but there
are a few enzymes, such as maltase (which acts on all α-glucosides) and
emulsin (which acts on all β-glucosides) which act catalytically upon groups
of considerable numbers of similar compounds.

Enzymes, like all other catalysts, act more energetically at increased
temperatures; but for each particular enzyme there is an "optimum
temperature," (usually between 40° and 65°) above which the destructive
effect of the temperature upon the enzyme itself more than offsets the
accelerating influence of the increased temperature. At still higher
temperatures (usually 80° to 100°) the enzymes are "killed," i.e., rendered
permanently inactive. All enzymes are "killed" by boiling the solutions in
which they are contained. Dry preparations of enzyme material can withstand
somewhat higher temperatures, for somewhat longer periods of time, than can
the same enzyme in moist condition or in solution. When an enzyme has once
been inactivated by heating, or "killed," it can never be restored to activity
again.

Enzymes are extremely sensitive to acids, bases, or salts, their activity being
often enormously enhanced or, in other cases, entirely inhibited, by the

presence in the reacting medium of very small amounts of free acids, or bases,
or even of certain neutral salts. For example, pepsin, the enzyme of the
stomach will act only in the presence of a slightly acid medium and is wholly
inactive in a mixture which contains even the slightest amount of free alkaline
material; while trypsin, the similar enzyme of the intestine, acts only under
alkaline conditions. Practically all enzymes are rendered inactive, but not
destroyed, by the presence of either acid or alkali in excess of N/10 strength.
Many will act only in the presence of small quantities of certain specific
neutral salts; while, on the other hand, other salts are powerful inhibitors of
enzyme action. Enzymes often differ from the protoplasm which secretes them
in their response to antiseptics, such as toluene, xylene, etc., which inhibit the
activity or growth of the cell, but have no effect upon the activity of the
enzymes which it contains.

THE CHEMICAL NATURE OF ENZYMES

Nothing is known with certainty concerning the chemical nature of
enzymes. Being colloidal in nature, they adsorb carbohydrates, proteins, fats,
etc., so that active enzyme preparations often respond to the characteristic tests
for these groups of substances; and many investigators have reported what
has, at first, seemed to be conclusive evidence that some particular enzyme
which they have studied is either a carbohydrate, a protein, or some other type
of organic compound. Later investigations have always shown, however, that
if the preparation in question be submitted to the digestive action of the
enzymes which hydrolyze the particular type of substances to which it is
supposed to belong, the material will lose its characteristic protein, or
carbohydrate, etc., properties, without losing its specific activity, thus clearly
indicating that the substance which responds to the characteristic tests for
some well-known type of organic compounds is present as an impurity and is
not the enzyme itself.

The present state of knowledge concerning the nature of enzymes seems to
indicate that, like the inorganic catalysts, they may vary widely in chemical
composition; and that their tremendous catalytic effects are due, in part at
least, to their colloidal nature. This will be better understood and appreciated
after the phenomena associated with the colloidal condition have been
considered (see the following Chapter).

NOMENCLATURE AND CLASSIFICATION

Since nothing is known of the chemical composition of enzymes, they can
only be studied by considering the effects which they produce. This is
reflected in the systems which have been adopted for their nomenclature and
classification.

As they were first supposed to be proteins, the earlier representatives of the
group were given characteristic names ending with the suffix in, similar to that
of the proteins. Since this idea has been found to be incorrect, however, a
system of nomenclature has been adopted which assigns to each enzyme the
name of the material upon which it acts, followed by the suffix ase. Thus,
cellulase is the enzyme which accelerates the hydrolysis of cellulose; glucase,
that acting upon glucose; amylase, that acting upon starch (amylum), etc.

The substance upon which the enzyme acts (or, strictly speaking, the
substance whose hydrolysis, oxidation, or other chemical change, is
catalytically affected by the enzyme) is called the substrate.

Most enzymes are catalysts for hydrolysis reactions and are, hence, classed
as hydrolytic in their action, and may be spoken of as "hydrolases." Those
which accelerate oxidation are called "oxidases"; while those that stimulate
reduction reactions are "reductases"; those that aid in the splitting off of

ammonia, or amino-acid groups, are "deaminases"; and those that aid in the
splitting off of CO2 from COOH groups are "carboxylases," etc.

The hydrolytic enzymes are further subdivided into the sucroclastic (sugar-
splitting), or sucrases; the lipoclastic (fat-splitting), or lipases; the esterases
(ester-splitting); proteoclastic (protein-splitting), or proteases; etc.

OCCURRENCE AND PREPARATION FOR STUDY

Enzymes are present in all living matter. In animal tissues, they occur in the
largest amounts in those glands or organs where active vital processes take
place, as in the brain, the digestive tract, blood, etc. In plants, they may be
found in all living cells, and are especially abundant in the seeds, where they
serve to render soluble and available to the young plant the stored food
materials. The enzymes of moulds, and other parasitic plants, are usually
extracellular in type, being secreted for the purpose of making the material of
the host plant available to the parasite. Extracellular enzymes are also
developed in seeds during germination, in order that the stored food material
of the endosperm may be rendered soluble and translocated into the tissues of
the growing seedling. But most other plant enzymes are intracellular in type.
Hence, in all preparations of plant enzymes for study, or for commercial use,
the first step in the process is, necessarily, a thorough rupturing of the cell-
walls of the plant material.

The rupturing of the cells may be accomplished in a variety of ways, as
follows: (1) mechanical disintegration, as by grinding in a mortar with sharp
sand; (2) freezing the material, by treatment with liquid air, then grinding; (3)
killing the cells by drying, by treatment with alcohol or acetone, then grinding
the mass in a paint mill with toluene; (4) killing the cells by chemicals
(sulfuric acid, 0.5 to 1.0 per cent, or other suitable agents) followed by
extraction with water; (5) autolysis, or self-digestion, in which the cells are

mixed with toluene or some other antiseptic which kills the cells without
injuring the enzymes, then the material is minced or ground up and suspended
in water containing the antiseptic, until the enzymes dissolve the cell-walls
and so escape into the liquid—this process being especially adapted to the
preparation of active extracts from yeasts, which contain the necessary cell-
wall dissolving enzymes to facilitate autolysis.

Enzymes may be separated out of the aqueous extract obtained from cells
ruptured by any of the above methods, by precipitation with alcohol, acetone,
or ether, in which they are insoluble; but if this is done, the precipitate must be
at once filtered off and rapidly washed and dried, as prolonged contact with
these precipitating agents greatly diminishes the activity of most enzymes. Or,
they may be adsorbed out of solution on gelatinous, or colloidal, materials,
like aluminium hydroxide, or various hydrated clays. If the dry preparations
obtained in any of these ways are contaminated by carbohydrates, proteins,
etc., these may be removed by treatment with suitable digesting enzymes
obtained from the saliva, gastric, and pancreatic juices, and the digested
impurities washed out with 60 to 80 per cent alcohol, leaving the enzyme
preparation in a purified but still active form.

In any study of the "strength," or possible catalytic effects, of an enzyme
preparation, it is necessary, first, to determine what particular reaction it
affects, by qualitative tests with various substrate materials, such as starch,
sugars, glucosides, proteins, etc., and then to determine quantitatively its
accelerating effect upon the reaction in question. The latter may be done by
measuring either the time required to carry a unit quantity of the substrate
material through any determined stage of chemical change, or the quantity of
the substrate which is changed in a unit period of time. It would not be
profitable to go into a detailed discussion here of the methods of making these
quantitative measurements of enzyme activity. Such discussions must
necessarily be left to special treatises on methods of study of enzyme action. It
may be said, however, that generally both the qualitative tests for, and the

quantitative measurements of, the accelerating influence of enzymes depend
upon the observation of some change in the physical properties of the
substrate material, such as the optical activity, electrical conductivity, or
viscosity, of its solution. In some cases, it is convenient to make an actual
quantitative determination of the amount of end-products produced in a given
time, as in the inversion of cane sugar, the hydrolysis of maltose, etc., but such
determinations necessarily involve the removal of some of the reaction
mixture for the purposes of the determinations, and are not, therefore, suitable
for the study of the progressive development of the reaction which is being
studied.

Enzymes are found in all parts of the animal organism and those which are
active in the digestion of food, the metabolism of digested material, the
coagulation of blood, etc., have been extensively studied. A discussion of these
animal enzymes would be out of place in such a text as this, however, and the
following list includes only enzymes which are known to occur in plant
tissues. These well-known enzymes will serve as examples of the several
general types which have thus far been isolated and studied.

Class and
Enzyme. Substrate. End-products. Found in.
Type.
I. Hydrolases
Glycerol and
(a) Esterases Lipase Fats Oily seeds
fatty acids

(b) Sucrase or Glucose and
Sucrose Yeasts
Carbohydrases invertase fructose
Maltose and all
Maltase Glucose, etc. Barley malt
α-glucosides
Dextrinase Dextrin Maltose Malt
Artichokes,
Inulase Inulin Fructose
etc.

Class and
Enzyme. Substrate. End-products. Found in.
Type.
Amylase or
Starch Maltose Malt etc.
diastase
Cellulase Cellulose Maltose Bacteria
Pectinase Pectose Arabinose Fruits
Mono- Nuts, seeds,
Cytase Hemi-celluloses
saccharides etc.

(c) Amygdalin and Almond
Emulsin Glucose, etc.
Glucosidases all β-glucosides kernels, etc.
Maltase α-glucosides Glucose, etc. Barley malt
Sulfur-containing Mustard
Myrosin Glucose, etc.
glucosides seeds
Rhamnus
Rhamnase Xanthorhamnin Rhamnose, etc.
spp.
Inosite and Bran coats
Phytase Phytin H3PO4 of seeds

(d) Proteases Erepsin Proteins Amino-acids Many plants
Papain Protein Amino-acids Papaws
Bromelin Protein Amino-acids Many plants
Proteins and
Nuclease Nucleo-proteins Many plants
nucleic acid
II. Oxidases
Hydrogen Water and Nearly all
(a) Catalases ........
peroxide oxygen plants
(b) Organic "Active" Nearly all
........
Peroxidases peroxides oxygen plants
Chromogens
Pigments Many plants
(c) Oxidases ........ Alcohols and
Acids Many plants
phenols
(d)
........ ........ ........ Many plants
Reductases

Class and
Enzyme. Substrate. End-products. Found in.
Type.
Ammonia and
III. Deaminases Urease Urea CO2
Guanase Guanine Xanthine
Adenase Adenine Hypoxanthine

IV.
Carboxylases
Aldehydes and
(a) ........ Keto-acids CO2
Amines and
(b) ........ Amino-acids CO2

V. Coagulation Coagulates pectic
Pectase ........ Fruits
enzymes bodies

VI.
Alcohol and
Fermentation Zymase Glucose, etc. Yeasts
CO2
enzymes
Lactic acid
Fatty acids Lactic acid Bacteria
ferment
Butyric
acid Fatty acids Butyric acid Bacteria
ferment

The above list includes only the more common and best-known plant
enzymes. It seems reasonable to suppose that for every individual type of
organic compound which may occur in general plant groups, or even in single
species, there is a corresponding enzyme available to affect its physiological
alterations. Indeed, new preparations of active enzymes from special types of

plants and new evidences of the existence of enzymes in various plant
organisms are continuously being reported.

A few of the most common specific representatives of individual groups of
enzymes may be briefly described, as follows:

Amylase (or diastase, as it was first named and is still commonly called) is
probably the most widely distributed enzyme of plants. It is found in
practically all bacteria and fungi; in practically all seeds (it has been found in
active form in seeds which were known to be over fifty years old); in all roots
and tubers; and in practically all leaves, where it is located in the stroma of the
chloroplasts.

It appears to exist in two modifications, known, respectively, as (a)
translocation diastase and (b) diastase of secretion. The first form is found in
the cells of ungerminated seeds, in leaves, shoots, etc. It remains in the cells
where reserve starch is stored and aids in the transformation of starch into
soluble materials for translocation from cell to cell. It is active at a lower
temperature than the second form, its optimum temperature being 45° to 50°.
The second form is secreted by the scutellum, and perhaps by the aleurone
cells, of germinating seeds, being produced by special glandular tissue. It aids
in the hydrolysis of the starch for the use of the growing embryo. Its optimum
temperature is 50° to 55°.

The activity of amylase is accelerated by the presence of small quantities of
neutral salts, especially by sodium chloride and disodium phosphate. It acts
best in neutral solutions, its activity being inhibited, although the enzyme itself
is not destroyed, by the presence of more than minute traces of free mineral
acid or alkali.

Sucrase (or invertase) is present in almost all species of yeasts, where it
serves to convert unfermentable sucrose into glucose and fructose, which are
readily fermentable. Invertase is also present in moulds and other

microorganisms; and in the buds, leaves, flowers, and rootlets of those higher
order plants which store their carbohydrate reserves in the form of sucrose. It
appears that sucrose, while easily soluble, is not readily translocated, or
utilized, by plants until after it has been hydrolyzed into its constituent
hexoses.

The optimum temperature for invertase is 50° to 54°; it is killed if heated, in
the moist condition, to 70°. Its activity is increased by the presence of small
amounts of free acids; but is inhibited by free alkalies.

Zymase is the active alcoholic fermentation enzyme of yeasts. It accelerates
the well-known reaction for the conversion of hexose sugars into alcohol and
carbon dioxide, namely,

C6H12O6 = 2C2H5OH + 2CO2.

Because of its scientific interest and industrial importance in the
fermentation industries, its action has been extensively studied. It acts only in
the presence of soluble phosphates and of a coenzyme (see below) which is
dialyzable and not destroyed, which is probably an organic ester of phosphoric
acid. The significance of the molecular configuration of the hexose sugars in
their susceptibility to action by zymase has already been discussed in detail
(see page 56).

The optimum temperature for zymase action is 28° to 30°. The enzyme is
killed by heating to 45° to 50° in solution, or to 85° if in dry preparation.

Proteases of the erepsin type, i.e., those which break proteins down to
amino-acids instead of only to the proteose or peptone stage, as is
characteristic of the enzymes of the trypsin type, are widely distributed in
plants. Except in the case of the two which occur in large amounts in certain
special fruits (papain in papaws, and bromelin in pineapples), they are very
difficult to prepare in pure form for study. In general, all proteolytic actions,

even when accelerated by active enzymes, proceed much more slowly than do
the hydrolyses of carbohydrates or fats. It seems that metabolic changes of the
complex protein molecules are much more difficult to bring about and take
place much more slowly than do those of the energy-producing types of
compounds.

The presence of proteolytic enzymes in most vegetative cells, and in seeds,
may be demonstrated, however, by studying the action of extracts of these
tissues upon soluble proteins. The best-known example of this type of
enzymes is the protease of yeast; but similar ones may be found in
germinating seeds. These vegetable proteases are usually most active in
neutral or only faintly alkaline solutions, and their activity is nearly always
inhibited by even traces of free acids.

Most laboratory studies of proteolytic enzymes are carried on with
preparations of the powerful members of this class of enzymes which are
found in the digestive tract of animals, namely, the pepsin of the gastric juice,
which acts in the acid medium, in the stomach, and the trypsin of the
pancreatic juice, which acts in the alkaline medium of the intestinal tract. But
even these powerful proteases require several hours for the transformation of
an amount of soluble albumin into its amino-acid constituents which is
equivalent to the amount of starch which is hydrolyzed to maltose by diastase
in a very few minutes.

Enzymes which govern oxidative changes, known respectively, as catalases
and oxidases, are almost universally present in plants. Catalase decomposes
peroxides, with the liberation of free oxygen. It is, therefore, necessary to the
final step in the process of photosynthesis, as elucidated by Usher and
Priestley (see page 26), and serves to prevent the destructive action of
hydrogen peroxide upon chlorophyll. The almost universal presence of
oxidases in plant tissues has been repeatedly demonstrated. They are present in
especially large amounts in tissues which are being acted upon by parasitic

fungi or are combating unfavorable conditions of growth. The oxidases, in
such cases, seem to be the agents by which the plant is able to stimulate its
metabolic activities to overcome the unfavorable environment for its normal
development.

In vegetables and fruits, the common browning, or blackening, of the
tissues when cut surfaces are exposed to the air has been demonstrated to be
due to the catalytic oxidation of the tannins or of certain amino-acids,
especially tyrosine, under the influence of the oxidases which are present in
the tissues. In fact, most pigmentation phenomena are due to changes in the
oxygen content of the chromogens of the cells of the plant, under the influence
of the oxidases which are present in the protoplasm of the cells in question.
Hence, the oxidases may be said to be the controlling agencies for both the
energy-absorbing activities and for respiration in plants.

THE NATURE OF ENZYME ACTION

The mechanism by which an enzyme accomplishes its catalytic effects has
been the object of extensive studies during recent years, especially since the
discovery by Büchner that enzymes could be isolated in solutions entirely free
from the disturbing influence of growing cells. Several theories concerning the
mode of this catalytic action have been advanced. The earliest and simplest of
these was that the enzyme simply creates an environment favorable for the
particular chemical reaction to take place, as by exposing large surfaces of the
substance in question to the action of the hydrolytic, or other effective, agent,
by means of surface adsorption of the substrate material on the colloidal
enzyme.

However, more recent investigations clearly indicate that there is an actual
combination between the substrate material and the enzyme, which
combination then breaks down with a resultant change in the substrate

material and a freeing of the enzyme for repeated recombination with
additional substrate, with the net result that the chemical change in the
substrate material is enormously accelerated. That such a combination
between substrate and enzyme actually exists has been demonstrated in two
different ways: (a) experimentally, by mixing together solutions of an enzyme
and of its substrate, each of which is filterable through paper or through a
porous clay filter, with the result that the active material in the combined
solutions will not pass through these same filters; and (b) mathematically, by a
study of the curves representing the reaction velocities of typical reactions
which are proceeding under the influence of an enzyme, which show that so
long as there is a large excess of substrate material present, the accelerating
influence of the catalyst is uniform over given successive periods of time, but
that when the quantity of substrate material becomes smaller than that which
permits the maximum combining power of the enzyme to be exercised, the
reaction velocity immediately slows up.

Again, the fact that the specificity of the action of an enzyme, i.e., the
limitation of the action of that enzyme to a specific single compound or group
of similar compounds, is definitely related to the molecular configuration of
the molecule of the substrate, as has been found to be true in all those cases
where the molecular configuration of the substrate material has been
established (see pages 56 to 58), is an added indication that there is some kind
of a union between the enzyme and the substrate as a first step in the catalytic
process.

As to the nature of this supposed combination of substrate and enzyme, two
theories are held. The first is that this union is in the form of an actual
molecular combination, or chemical compound, and the other is that it is a
purely physical, or colloidal complex. The latter view has by far the greater
weight of theoretical and experimental evidence in its support. The relation of
electrolytes to the catalytic effect of enzymes, the appearance of the reacting
masses under the ultramicroscope, and the effect of heat upon the reacting

mixtures, all point to the conclusion that the phenomenon is colloidal rather
than molecular in character. This view also makes the remarkable catalytic
effects which take place in living protoplasm, which undoubtedly exists in the
colloidal condition, much more easily understood. This phase of the matter
will be much more apparent after the chapter dealing with the physical
chemistry of the protoplasm has been studied.

A further indication that the mechanism of enzyme activity is colloidal in
character lies in the fact that, so far as is known, all reactions which are
catalyzed by specific enzymes are reversible and the same enzyme will
accelerate the velocity of the reaction in either direction, the direction in which
the reaction goes being determined by the conditions surrounding the reacting
material at the time. It was formerly supposed that enzymes catalyze only
decomposition reactions and that the synthetic reactions of living tissues are
produced by means of some other force or agency. This view supported the
idea of a chemical union of the enzyme with the substrate which, when it
breaks down, breaks the molecule of the substrate material into some simpler
form, or forms. But it is now known that the reaction which is influenced by
the enzyme will be catalyzed in either direction by the specific enzyme which
"fits" the particular substrate material at every point of its molecular
configuration, as the glove fits the hand. The contrast between this fitting of
the enzyme to the entire configuration of the molecule, and the union at a
single point or group which is characteristic of chemical linkages, is apparent.
As examples of the synthetic action of the same enzyme which, under other
conditions, accelerates the decomposition of the same material, there may be
cited the demonstrated synthesis of isomaltose from glucose by maltase; the
production of ethyl butyrate from alcohol and butyric acid; and the synthetic
production of artificial fats, by the aid of the pancreatic lipase; and the
apparent synthesis of a protein from the same amino-acids which may be
obtained from it by hydrolysis under the influence of the same protease, but
under different environmental conditions.

ACTIVATORS AND INHIBITORS

The activity of enzymes is strongly influenced by the presence in the
solution of other bodies, usually, although not always, electrolytes. This is
probably due, in most cases at least, to the action of the electrolyte upon the
colloidal condition of the enzyme. All enzymes do not respond alike to the
action of the same electrolyte, however. The activity of certain enzymes is
enormously increased by the presence of a small amount of acid; while the
action of another may be absolutely inhibited by the same acid in the same
concentration. Thus, the activity of the amylase found in the endosperm of
many seeds is instantly stopped by adding to the solution enough sulfuric acid
to make it two-hundredth normal in strength; while the same concentration of
acid actually accelerates the activity of some of the proteases.

Formaldehyde, hydrocyanic acid, and soluble fluorides usually inhibit both
the activity of a cell and of the enzymes which it contains; while other
antiseptics, such as toluene, xylene, etc., prevent the growth of the cell, or
organism, without interfering with the activity of the enzymes which may be
present. By the use of this latter type of antiseptics, it is possible to distinguish
between chemical changes which are involved in the actual development of a
cell and those which can be brought about in other media by means of the
enzymes which are contained in the cell.

Any substance which increases the catalytic activity of an enzyme is known
as an "accelerator," or "activator"; while one which prevents this activity is
called an "inhibitor," or "paralyzer."

A type of accelerating influence quite different from that of electrolytes is
found in the effect of certain amino-acids upon enzyme action. The influence
of small amounts of asparagine in enormously increasing the hydrolytic effect
of amylase is an example. There is no known explanation for this type of
activation of the enzyme.

The influence of activators, or inhibitors, in providing favorable or
unfavorable conditions for the action of an enzyme, should not be confused
with the relation to the enzyme itself of what are known as "coenzymes" and
"antienzymes," discussed in the following paragraph.

COENZYMES AND ANTIENZYMES

In the cases of many enzymes of animal tissues, it has been found that they
are absolutely inactive unless accompanied by some other substance which is
normally present in the gland, or protoplasm, which secretes them. Thus, the
bile salts are absolutely necessary to the activity of trypsin, in its characteristic
protein-splitting action. Such substances are known as "coenzymes." They can
usually be separated from their corresponding enzymes by dialysis, the
coenzyme passing through the parchment membrane. Such coenzymes are not
killed by boiling the dialyzate, and the activity of the enzyme is restored by
adding the boiled dialyzate to the liquid which remains within the dialyzer.

The best known example of a coenzyme in plant tissues is in connection
with the activity of the zymase of yeast cells. If yeast juice be filtered through
a gelatin filter, the colloidal enzymes which are left behind are entirely
inactive in producing fermentation, but may be restored to activity again by
mixing with the filtrate. An examination of this filtrate, which contains the
coenzyme for zymase, shows that it contains soluble phosphates and some
other substance whose exact nature has not yet been determined, both of
which are necessary to the activity of the zymase. The phosphates seem to
enter into some definite chemical combination with the substrate sugars, while
the other coenzyme seems to be necessary in order to make possible the final
breaking down of the sugar-phosphate complex by the zymase. This
phenomenon of coenzyme relationship is not very frequently observed in plant
enzyme studies, probably because the coenzyme (if there be such, in the case

which is under observation) usually accompanies the enzyme itself through
the various processes of extraction and purification of the material for study.
However, care must be taken in all cases when dialysis is employed, to see
that a possible coenzyme is not separated from an otherwise active
preparation.

An entirely different type of phenomenon is that exhibited by
"antienzymes." These are found in the various intestinal worms which live in
the digestive tracts of animals; and prevent the digestive action of the enzymes
of the stomach and intestines upon these worms. Probably similar
"antienzymes" are located in the mucous linings of the intestinal tract itself,
and serve to prevent the auto-digestion of these organs by the active enzymes
with which they are almost continually in contact.

The difference between an antienzyme, which protects material which
would otherwise be subject to the attack of an enzyme, and an inhibitor, which
renders the enzyme itself inactive, is apparent.

So far as is known, however, no such substances as antienzymes are present
in plant tissues; although the question as to why the proteoclastic enzymes
which are elaborated by a given mass of protoplasm do not attack the
protoplasm itself, might well be raised.

ZYMOGENS

It is apparent that, since enzymes are produced by protoplasm for the
special needs of any given moment or stage of development, there must be a
preliminary stage, or condition, in which they do not exert their characteristic
catalytic effect. When in this stage, the compound is known as "proenzyme,"
or "zymogen." In this stage, it is inactive, but can be made to exhibit its
catalytic effect, usually by bringing it into contact with a suitable activator.

When once so activated, however, it cannot be returned again to the inactive
state.

This phenomenon has been studied in connection with the zymogens of the
digestive proteases, pepsin and trypsin. Trypsinogen may be rendered active
by contact with either calcium salts or with another substance (apparently
itself an enzyme) known as enterokinase, which is secreted in the intestinal
tract.

Similarly, proenzymes have been reported as occurring in numerous plant
tissues. These proenzymes are believed to be present in the plant cells in the
form of definite characteristic granules, which may be observed under the
microscope, and which disappear when the enzyme becomes active. Thus,
"proinulase" has been reported as occurring in artichoke tubers: "prolipase," in
castor beans; "proinvertase," in several species of fungi; and, probably,
"prooxidase," in tobacco leaves. In the case of the last-named zymogen, it has
been observed that after the zymogen has been once activated, as in response
to the need for increased activity due to the entrance of the germs of certain
leaf-diseases, it can once again produce a second supply of the enzyme, but
the process cannot again be repeated.

Calcium salts, or very dilute acids, are usually energetic activators of
proenzymes.

PHYSIOLOGICAL USES OF ENZYMES

There can be no doubt that enzymes exert a tremendously important
influence in vital phenomena, by determining the rate at which the chemical
changes which are involved in these phenomena shall proceed. Since they do
not initiate reactions, and since they may catalyze reversible reactions in either
direction, it cannot be said that they determine the type of reactions which will

take place in any given mass of protoplasm; but, undoubtedly, they do exert a
determining influence upon the rate at which the reaction will proceed, after
the protoplasmic activity has determined the direction in which it shall go.

Without the intervention of these catalyzing agents, it would be impossible
for reactions between these non-ionized organic components of the cell
contents to come to completion with anything like the marvelous rapidity with
which these changes must take place in order to permit the organism to grow,
to perform its necessary vital functions, or to adjust itself to the changes in its
environmental conditions.

Since the number of different reactions which take place within a living cell
is very great, and since these chemical changes are extremely variable in type,
it follows that the number of different enzymes which must exist in either a
plant or an animal organism is likewise very large. For example, fourteen
different enzymes have been isolated from the digestive system, and at least
sixteen from the liver, of animals. They are universally present in living
protoplasm of every kind, from the most minute bacterium to the largest forest
trees, in the plant kingdom; and from the amœba to the whale, in animals.

While there is a great variety of enzymes which may be produced by a
single individual organism, the same enzyme may be found in the greatest
variety of organisms; as, for example, the protease trypsin, which has been
found in several species of bacteria, in the carnivorous plant known as "Venus'
Fly Trap," and in the human pancreas, as well as that of all other animals.

FURTHER STUDIES NEEDED

From the discussions which have been presented in this chapter, it is
apparent that the enzymes play a tremendously important part in vital

phenomena, by controlling the rate at which the biochemical reactions take
place in the cells of the living organism.

The means by which the protoplasm elaborates these all-important chemical
compounds are as yet absolutely unknown. Even the nature of the enzymes
themselves is still a matter of speculation and study. Much intensive study is
needed and should be given to these matters, for the purpose of elucidating the
methods by which the enzymes accomplish their remarkable catalytic effects,
and, if possible, the actual chemical nature of the enzymes themselves. It is
conceivable, of course, that if the latter object of these studies should ever be
reached, it might be possible to synthetize enzymes artificially, and so to
develop a means for the artificial duplication of the synthesis of organic
compounds with the same velocity that this is done in the plant cells. Such a
result would have a scientific interest fully as great as did Wöhler's artificial
synthesis of urea, which proved that there is no essential difference in
character between the compounds which are the products of living organisms
and those which are produced in the laboratory; and, at the same time, might
have an immensely more important practical bearing, since it would lead the
way to the artificial production of the carbohydrates, proteins, fats, etc., for
which we are now dependent upon plant growth as the source of these
materials for use as human food.

References

Bayliss, W. M.—"The Nature of Enzyme Action," 186 pages,
Monographs on Biochemistry, London, 1919 (4th ed.).

Euler, H., trans. by Pope, T. H.—"General Chemistry of the
Enzymes," 319 pages, 7 figs., New York, 1912.

Effront, J., trans. by Prescott, S. C.—"Enzymes and their
Application,—Enzymes of the Carbohydrates," 335 pages, New
York, 1902.

Effront, J., trans. by Prescott, S. C.—"Biochemical Catalysts in
Life and Industry—Proteolytic Enzymes," 763 pages, New York,
1917.

Green, J. R.—"The Soluble Ferments and Fermentation," 512
pages, Cambridge, 1901, (2d ed.).

Grus, J.—"Biologie und Kapillaranalyse der Enzyme," 227 pages,
58 figs., 3 plates, Berlin, 1912.

Harden, A.—"Alcoholic Fermentation," 156 pages, 8 figs.,
Monographs on Biochemistry, London, 1914.

Plimmer, R. H. A.—"The Chemical Changes and Products
Resulting from Fermentations," 184 pages, London, 1903.

Oppenheimer, C., trans. by Mitchell, C. A.—"Ferments and their
Actions," 343 pages, London, 1901.

CHAPTER XV

THE COLLOIDAL CONDITION

Reference has frequently been made, in preceding chapters, to the fact that
proteins, enzymes, lipoids, etc., exist in the protoplasm of plants and animals
in the colloidal condition. The properties and uses of these compounds by
plants depend so much upon this fact that, before proceeding to the
consideration of the actual physical chemistry of protoplasm itself, it will be
appropriate and profitable to give some attention to the nature and significance
of the colloidal condition of matter and of some of the phenomena which grow
out of it.

Every discussion of the colloidal condition in general properly begins with
reference to the work of the English physicist, Thomas Graham, who carried
on his investigations of the so-called "colloids" through a period of forty
years, beginning with 1851. His most important results were published,
however, from 1861 to 1864. Graham studied the diffusibility of substances in
solution through the parchment membrane of a simple dialyzer. As a result of
his earlier investigations, he divided all the chemical compounds which were
known to him into two groups, which he called "crystalloids" and "colloids,"
respectively, the first including those substances which readily diffused
through the parchment membrane and the second those which diffused only
very slowly or not at all. He at first thought that crystalloids are always

inorganic compounds, while colloids are of organic origin. He soon learned,
however, that this distinction in behavior is not always related to the organic
or inorganic nature of the compound. He further discovered that the same
individual chemical element or compound may exist sometimes in
crystalloidal, and sometimes in colloidal, form. This latter discovery led to the
conclusion that diffusibility depends upon the condition, rather than upon the
nature, of the material under observation.

As a result of the long series of investigations which were stimulated by
Graham's work, the modern conception is that diffusibility is a condition of
matter when in minute subdivision, or in solution, in some liquid, as
contrasted with its state, or condition, when existing alone. That is, the state of
a substance may be either gaseous, liquid, or solid; and its condition when in
solution may be either crystalloidal or colloidal. Substances which are in
crystalloidal form, in true solution, exist there in molecular or ionized
condition; but, as will be pointed out below, when in the colloidal condition
they exist in aggregates which are somewhat larger than molecules, but not
large enough to be visible as individual particles under the ordinary
microscope, even under the highest magnification which has yet been
obtained. Colloidal particles are, however, generally visible under the
Zigmondy "ultramicroscope." (See below.)

The use of the word "colloid" as a noun, or as the name for a substance
which is in the colloidal condition, is of the same nature as the use of the
words "gas," "liquid," and "solid," in such statements as "ice is a solid," "water
is a liquid," or "steam is a gas," etc.; i.e., the noun represents a state or
condition rather than an actual object or thing. Hence, the expression
"enzymes are colloids," means only that enzymes exist in the colloidal
condition, and not that enzymes represent a definite type of substances having
the group name "colloids."

THE COLLOIDAL CONDITION A DISPERSION
PHENOMENON

When one substance is distributed through the mass of another substance,
the mixture is said to be a "two-phase system," composed of the dispersed
phase, or substance, and the dispersion medium, or continuous phase, through
which the other substance is distributed. The following examples illustrate the
possibilities of such two-phase systems:

1. Dispersion medium a gas.
a. Disperse phase a liquid—mist in the air.
b. Disperse phase a solid—smoke or dust in air.

2. Dispersion medium a liquid.
a. Disperse phase a gas—foams.
b. Disperse phase a liquid—emulsions.
c. Disperse phase a solid—suspensions.

3. Dispersion medium a solid.
a. Disperse phase a gas—solid foams, pumice stone, etc.
b. Disperse phase a liquid—liquid inclusions in minerals.
c. Disperse phase a solid—alloys, colored glass, etc.

Although the same general principles of physical chemistry apply to all
two-phase systems, the term "colloidal condition" is commonly used only in
connection with a particular type of dispersions, in which the dispersion
medium is a liquid and the dispersed material is either a solid or a liquid.

Thorough and careful studies have shown that when a solid or a liquid is
introduced into another liquid, and becomes dispersed or distributed through
it, the mixture may be either a true solution, a colloidal solution, or a
mechanical suspension. The characteristic differences between these three
conditions may be tabulated as follows: although the significance of some of

the phrases used will not be apparent until the phenomena in question have
been considered in some detail.

True Solutions. Colloidal Solutions. Suspensions.
(a) Particles of the disperse
phase are:
In molecular In mechanical
In colloidal subdivision
subdivision subdivision
Visible under
Visible under
Invisible microscope or to
"ultrascope"
naked eye
Less than 1µµ in Greater than 1µ in
1µµ to 1µ in diameter
diameter [6] diameter
Do not pass through
In molecular motion In Brownian movement
filters or parchment
Pass through filters and Pass through filters but In gravitational
parchment membranes not through parchment movement

(b) The system exhibits:
High osmotic pressur Low osmotic pressure No osmotic pressure
Transparency "Tyndall phenomenon" Is generally opaque
No gel-formation Forms gels No gel-formation

[6 1µ is one-thousandth of a millimeter; 1µµ is one-thousandth of a µ, or one millionth of a
] millimeter.

It is recognized by all students of these matters that it is not possible to draw
a sharp dividing line between these three types of conditions, and that they
shade into each other, in many cases; but in general it may be said that a
colloidal solution is one in which the dispersed particles are usually between
5µµ and 200µµ in diameter, are difficultly or not at all diffusible through the
membrane of a simple dialyzer, cannot be filtered out of solution, do not settle

out under the action of gravitation, and are visible only under the
"ultramicroscope"; and one which has certain peculiar optical, osmotic, and
other physical and chemical properties. Since colloidal particles are very
minute in size, they possess very large relative surface areas as compared with
their total mass or volume, very high surface tension, and a relatively high
surface energy as compared with their total, or molecular, energy. These
properties bring into play, in a substance which is in the colloidal condition, in
a remarkable degree, all the phenomena which are associated with surface
boundaries between solids and liquids, liquids and gases, etc.

The properties arising out of the colloidal condition are of such tremendous
importance in connection with the vital phenomena exhibited by cell
protoplasm that it is necessary to give some detailed consideration to them
here. Many large volumes dealing with this condition of matter have been
written, and it is very difficult to condense even the most important facts
concerning it into a few pages, but an attempt has been made to present in this
brief summary the most essential facts and principles involved in the colloidal
phenomena.

NOMENCLATURE AND CLASSIFICATION

Colloidal mixtures may exist in two different forms: one, in which the
mixture is fluid and mobile, like a true solution, is known as a "sol"; and the
other, which is a semi-solid, or jelly-like, form, is known as a "gel." Sols may
be easily converted (or "set") into gels, by changes of temperature or of the
electrolyte content, or by changes in the concentration of the mixture, etc., and
in most cases gels can be converted again into sols. In some cases, however,
gel-formation is irreversible, the gels are permanent and cannot be changed
back again into sols by any known change in environmental conditions.

Depending upon whether the liquid dispersion medium is water, alcohol,
ether, etc., sols are known as "hydrosols," "alcosols," "ethersols," etc.; and gels
as "hydrogels," "alcogels," etc.

Sols in which the disperse phase is a solid are known as "suspensoids";
while those in which it is a liquid are "emulsoids." Thus, sols of most
inorganic compounds, of dextrin, gelatin, and (probably) of casein, etc., are
suspensoids; while sols of egg-albumin, of oils, etc., are emulsoids. The
classification of these substances into suspensoids and emulsoids is, however,
more a matter of convenience than of real difference in composition, since it is
practically impossible to say whether many of the organic substances which
normally exist in colloidal form are themselves liquids or solids, when in the
non-dispersed form.

CONDITIONS NECESSARY TO THE FORMATION
OF SOLS

Suspensoids differ from mechanical suspension of solids in a liquid in that
in the latter the solid particles settle toward the bottom of the mixture, because
of the effect of the attraction of gravity upon them. The rate at which such
particles settle depends upon the size and density of the particle and the
viscosity of the liquid, and can be roughly calculated from the formula for
Stokes' law for the rate of falling of a spherical body in a liquid. This formula
is

2r2(s - s´)g
V = —————
9n

V = velocity of the falling body, in millimeters per second;
r = radius of the particle, in millimeters;
s = specific gravity of the solid;