BIOLOGY
LIBRARY

G

PRACTICAL
PLANT BIOCHEMISTRY

CAMBRIDGE UNIVERSITY PRESS

C. F. CLAY, MANAGER
LONDON : FETTER LANE E. C. 4

LONDON : H. K. LEWIS & CO., LTD.,
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ALL RIGHTS RESERVED

PRACTICAL
PLANT BIOCHEMISTRY

BY

MURIEL WHELDALE ONSLOW

M

Formerly Fellow of Newnham College, Cambridge, and Research Student at

the John Innes Horticultural Institution, Merton, Surrey.

Author of The Anthocjanin Pigments of Plants.

CAMBRIDGE

AT THE UNIVERSITY PRESS
1920

. •

PREFACE

THIS book is intended primarily for students of Botany. Such a
student's knowledge of plant products is usually obtained, on the
one hand, from Organic Chemistry, on the other hand, from Plant
Physiology; between these two standpoints there is a gap, which, it is
hoped, the following pages may help to fill. It is essentially a text-book
for practical work, an aspect of Plant Biochemistry which has received up
to the present time very little consideration in teaching. A number of
experiments have been devised and have been actually tested in practical
classes. These experiments should enable a student to extract from the
plant itself the chemical compounds of which it is constituted, and to learn
something of their properties. An elementary knowledge of Organic
Chemistry on the part of the student has been assumed, as it appeared
superfluous to incorporate the material which has already been so amply
presented in innumerable text-books.

My sincerest thanks are due to Dr F. F. Blackman, F.R.S., for criticism
and many suggestions throughout the writing of the book. I am further
indebted to Mr H. Raistrick, M.A., for help in various ways, especially in
reading the proof-sheets. I wish, in addition, to express my gratitude to
Professor F. G. Hopkins, F.R.S., for the great interest he has always shown
in the subject and for his kind and stimulating advice in connexion with
the scheme of teaching presented in the following pages.

M. W. O.

CAMBRIDGE,
February, 1920.

436986

CONTENTS

CHAP. PAQE

I. INTRODUCTION . <-.

II. THE COLLOIDAL STATE . . . . - 10

III. ENZYME ACTION . . • • . • 1L

IV. CARBON ASSIMILATION .... 26

V. CARBOHYDRATES AND THEIR HYDROLYZING

ENZYMES . .\ . . • • -41

VI. THE FATS AND LIPASES ... 79

VII. AROMATIC COMPOUNDS AND OXIDIZING EN-
ZYMES . '*- - 8?

VIII. THE PROTEINS AND PROTEASES . . . .118

IX. GLUCOSIDES AND GLUCOSIDE-SPLITTING EN-
ZYMES .... -142

X. THE PLANT BASES . . . . . - .154
INDEX .... 168

CHAPTER I

INTRODUCTION

This chapter should be re-read after the remaining chapters have been studied.

ALL plants are made up of a complex organized mixture of chemical
substances, both organic and inorganic. As a preliminary to the study
of plant chemistry, the student should realize that the chemical
compounds which make up the living plant may be approximately
grouped into the four following classes. Thus, in later chapters, when
reference is made to any plant product, it will be understood, broadly
, speaking, to which class it belongs, and what relationship it bears to
other chemical compounds.

The main classes may be enumerated as follows :

(1) Carbohydrates. These contain only carbon, hydrogen and oxygen.
The simplest members are the sugars, which are aldehydes and
ketones of polyhydric alcohols of the methane series of hydrocarbons.
The more complex carbohydrates, such as starch, cellulose, dextrins,
gums and mucilages, are condensation products of the simpler sugars.
The sugars are found in solution in the cell-sap of living cells throughout
the plant. Cellulose, in the form of cell-walls, constitutes an important
part of the structure of the plant, and starch is one of the most widely
distributed solid "reserve materials."

(2) Fats. These also contain only carbon, hydrogen and oxygen.
Chemically they are glycerides, that is glycerol esters of fatty acids.
These acids are derived from two series of hydrocarbons, i.e. the
methane series and the olefine series, and they usually contain a large
number of carbon atoms. The fats occur as globules deposited in the
cells, especially in the tissues of seeds where they form reserve materials,
though they also occur in other parts of plants. The acid components of
the fats are also found in the free state.

The carbohydrates and the fats both belong to the aliphatic series of
organic compounds, that is to the series in which the carbon atoms are
united in chains.

(3) Aromatic compounds. These are characterized by having the
carbon atoms united in a ring, as in benzene. They may contain more
than one carbon ring, and, moreover, aliphatic groupings may be attached

o. 1

INTRODUCTION [CH.

<• to the carbon ring as side-chains. The number of aromatic substances
is very great, and every plant contains representatives of the class.
Some members are widely distributed; others, as far as we know, are
restricted in their distribution, and may be peculiar to an order, a genus
or even a species. This class contains the various phenols, i.e. hydroxy-
derivatives of benzene, such as phloroglucin; and the corresponding acids,
i.e. hydroxy-derivatives of benzoic acid, such as gallic and protocatechuic
acids. Just as in the case of the carbohydrates, where simpler compounds
may become more complex by condensation, so the aromatic acids,
aldehydes, and their derivatives may be condensed to form more complex
substances, such as tannins. Other members containing more than one
benzene ring are the water-soluble yellow, red, purple and blue pigments
of plants, the yellow being hydroxy-flavones and flavonols, the remainder
V anthocyanins. The simplest aromatics occur in true solution in the
cell-sap throughout the plant, and the more complex ones exist in the
colloidal state.

Another section of aromatics is represented by the "essential oils."
They are heterogeneous in chemical composition, though they are
chiefly represented by the hydrocarbons of the terpene series. They also
include various alcohols, aldehydes and ketones. They have no relation-
ship with the true fats, but are responsible for many of the well-known
scents of plants ; as examples may be mentioned limonene in lemons,
pinene in Conifers, borneol in Thyme and Rosemary, menthol in
Peppermint and camphor in the Camphor tree (Laurus Camphora).

(4) Proteins. This large class contains substances which are in many
cases built up of groupings from both the aliphatic and aromatic series.
It includes not only the proteins but also their simpler derivatives, the
albumoses, peptones and polypeptides. In this case, as before, the simplest
derivatives, known as the amino-acids, are synthesized by condensation
to form the polypeptides, peptones, albumoses and proteins, in a series
of increasing complexity. The amino-acids are compounds, either of
the aliphatic or aromatic series, in which one or more hydrogen atoms
are replaced by the radicle NH2. They are soluble and crystalline,
but after condensing together, the final product, the protein, only exists
in either the solid or the colloidal state. Proteins, in the latter condition,
constitute the bulk of the complex material, protoplasm ; in the solid
state, in the form of grains and granules, they occur as reserve material
in the cell.

The above-mentioned classes will be dealt with in greater detail in
the later chapters. There are, of course, a large number of other

i] INTRODUCTION 3

substances in the plant, some of which may be included with the above,
while others form additional small classes, as for instance, the
alkaloids : others, again, bear no close relationship to any class, such as
the all-important pigment, chlorophyll.

In order to appreciate the subject of plant chemistry, the plant,
which is familiar as a botanical entity, must be interpreted in chemical
terms. The principal classes of the more essential and widely distri-
buted compounds found in plants have already been indicated on the
broadest basis, so that they may now be referred to without additional
comment.

From the botanical point of view, the plant may be regarded as a
structure composed of many living protoplasmic units enclosed in cell-
walls and combined together to form tissues. There are also certain
tissues, known as dead tissues, which assist in giving rigidity to the
plant. All these structural elements can be translated into terms of
chemical compounds.

One of the chemical processes most frequently met with in the plant
is that of synthesis by condensation, with elimination of water, of large
complex molecules from smaller and simpler molecules. The formation
of cellulose, for instance, is a case in point. Cellulose has the composition
(C6H10O5)n and, on hydrolysis with dilute acids, it yields glucose as a final
product. Hence it is concluded that the complex molecule of cellulose is
built up from the simpler carbohydrate by condensation. The synthesis
of proteins from ammo-acids affords another example. These acids
contain either an aliphatic or aromatic nucleus (let it be R), and one or
more carboxyl and arnino groups. Condensation takes place in the plant,
with elimination of water, according to the following scheme :

Ri Rii Riii R*

III I

NH2 CH— CO OH H NH CH— CO|OH HINH CH— CO;OH H;NH • CH— COOH

The products of such condensation, the proteins, vary among them-
selves according to the number and kind of ammo-acids which take part
in the synthesis.

Two important results arise from this process. First, the substances
formed by condensation have molecules of a very large size ; secondly,
whereas the simple compounds, sugars and amino-acids, are soluble,
crystalline and diffusible, the condensation products are either insoluble,
e.g. cellulose, or exist in the colloidal state, as is the case of many proteins j
and other plant constituents. As these very large molecules do not dialyze,'

4 INTRODUCTION [CH.

1 they remain where they are synthesized, and build up the solid
^structure of the plant, as for instance, the cell-walls.

Matter in the colloidal state is of very great importance in the plant
and is probably responsible for many of the properties of "living"
material. Thus it will not be out of place, though it will be referred to
again in a later chapter, to make at this point a few remarks on the
colloidal state. It has been known for some time that certain metals,
e.g. gold and silver, and also certain metallic hydroxides and sulphides,
e.g. ferric hydroxide and arsenious sulphide, though insoluble in water-
under ordinary conditions, can, by special methods, be obtained as
solutions which are clear to the unaided vision. Such solutions are
termed colloidal. Investigation has shown that the matter is not present
in true solution, but in a very finely divided state, i.e. as particles many
times larger than simple molecules, but smaller than any particles obtain-
able by mechanical means of division. Such solutions are known as
artificial colloidal solutions, but there are a number of organic substances,
with very large molecules, such as proteins, starch, gums, agar, etc., which
at once dissolve in water giving colloidal solutions. The main feature of
the colloidal state is that the system consists of two phases, or conditions
of matter. In the case of the artificial colloidal solutions first mentioned,
one state is solid, the gold particles; the other state is liquid, the water.
The solid is known as the dispersed phase, and the water as the continuous
phase, and such colloidal solutions are termed suspensoids. In the case
of proteins, starch, etc., both phases are liquid: the dispersed phase,
a concentrated solution of protein, etc.; the continuous phase, a
dilute solution of protein, etc. Such colloidal solutions are known as
emulsoids.

An important point in connexion with the colloidal state is that the
molecules, or aggregates of molecules, forming the dispersed phase are
so large that they exhibit some of the phenomena of surface energy,
^ electrical charge, etc., associated with matter in mass. These properties
come to be of considerable importance, when we consider how large
a surface is presented by matter in this state in comparison with its
mass.

A material in the plant upon which much interest naturally centres
is the protoplasm and its nucleus. It has been shown that the protoplasm
consists, chemically, largely of proteins in the colloidal state. It is itself
a liquid, and embedded in it are substances of various chemical constitu-
tion, in the form of granules of solid matter and also liquid globules.
Numerous chemical reactions are continually taking place in the

i] INTRODUCTION 5

protoplasm throughout the cell, and since many of these reactions can
take place both simultaneously and independently, the protoplasm must
have some form of organized structure. Though many phenomena of " life "
may be accounted for by the physical and chemical properties of such
substances as proteins, it is impossible to say, with our present knowledge,
how far all " living " phenomena may yet be explained in this way.

Some of the main lines of metabolic syntheses which take place in
the plant will next be considered. A fundamental fact which should be
borne in mind is that the green plant synthesizes all the complex
materials of which it is composed from the simple compounds, carbon
dioxide, water and certain inorganic salts. The most important factor, }
perhaps, which figures in plant metabolism, is chlorophyll. The green
pigments of chlorophyll are esters of complex organic acids containing
the elements carbon, hydrogen, oxygen, nitrogen and magnesium. They
have the remarkable power of absorbing the radiant energy of the sun's
rays and of transforming it into chemical energy, by means of which
carbon dioxide and water are combined to form some organic compound,
possibly formaldehyde, from which a simple carbohydrate is readily
synthesized.

If now the initial and final products of carbon assimilation be
considered in detail, it will be seen that the process is one of reduction :

6CO2+6H2O = C6H12O6 + 6O2.

This is confirmed by the fact that oxygen is evolved in the process.
Moreover, the plant accumulates a store of energy, since the final
product, the carbohydrate, has a higher potential energy than the system,
water and carbon dioxide. Hence carbon assimilation, in addition to
providing a basis of organic material as a starting-point for all the main
metabolic functions, also provides a source of chemical energy by means
of which reactions in other directions are brought about.

The setting free of this accumulated energy constitutes the process
of respiration, which is, in reality, an oxidation of carbohydrate taking
place in tissues throughout the plant. It is the converse of carbon
assimilation, in that oxygen is absorbed and carbon dioxide and water
are formed. Thus these two processes, both so fundamental and essential
to the metabolism of the green plant, are constantly taking place side
by side in the same cell.

The first-formed carbohydrate, which is probably a hexose, is
condensed in the plant, on the general lines we have previously indicated,
to form more complex disaccharides and polysaccharides, such as maltose,

6 INTRODUCTION [CH.

Cane-sugar, starch, cellulose, etc. Some of these products, such as the
disaccharides, form true solutions and may be present in the cell-sap ;
others, such as cellulose and starch, are present in the solid state, though
they contain considerable quantities of water. Others, again, such as
dextrin and gum, are present in the colloidal state. Thus, given an
initial carbohydrate and a source of energy, we may proceed to indicate
the other main lines of syntheses in the plant.

The next most important line of syntheses is probably that which
gives rise to the nitrogen-containing constituents of the plant. Nitrogen
is absorbed by the green plant in the form of nitrates and ammonium
salts, but the processes which lead to the synthesis of some of the
V j simplest nitrogen- containing compounds, such as the amino-acids, are still
very obscure. Aliphatic and aromatic acids of various kinds are
abundantly present in the tissues, but the reactions by which the NH2
groups are introduced are by no means clear. There is little doubt,
however, that once the amino-acids are formed, condensation takes place
as already indicated, and more complex molecules, termed polypeptides,
arise. Such polypeptides have now been synthesized artificially by the
condensation of amino-acids. From the polypeptides, by further stages
of condensation, the albumoses, peptones, and finally proteins are
produced.

Another line of syntheses is that which leads to the production of
the fats and allied substances. The fats are mainly glycerides of acids
of the methane and olefine series, such as butyric, palmitic and oleic
acids. Like ail other plant products the fa,ts must either directly or
indirectly arise from the carbohydrates. There is evidence that the
origin is fairly direct, as, for instance, in fatty seeds when the fats take
the place of sugars in ripening. The sugars, as we know, are aldehydes
of the polyhydric alcohols of the methane series. It has been suggested,
though the actual stages have not been ascertained, that by various
oxidation and reduction processes, the sugars yield fatty acid residues
which then condense to form the fatty acids of high molecular weights
present in fats. By a converse process, the fats, especially when they
are stored as reserve materials in seeds, are broken up, and sugars are
again formed which pass to other parts of the germinating seedling, and
are there used in other synthetic processes.

A third main line of syntheses is that which gives rise to the
aromatics of the plant. Since no ring compound is absorbed by the green
plant, it follows that by some process the aliphatic structure must be
transformed into the aromatic. Thus, for instance, the trihydric phenol,

i] INTRODUCTION 7

phloroglucin, might at some stage be formed from a hexose by conversion
of the aliphatic chain into a closed ring :

OH H OH OH

OHC— C— C— C— C— CH-,OH — 3H.,O = CO— CH2— CO— CH2— CO— CH.>
H OH H H 1 __ _ __ j

Glucose

H2 H

A /c\

OC CO HOC COH

I = II i

H.,C CHo HC CH

O OH

Phloroglucin

There is evidence that aromatic compounds, such as phloroglucin,
tannins, flavones and anthocyanins are synthesized in the leaves, and
that sugar-feeding, by floating leaves in sugar solutions, leads to the
increase of aromatics in the tissues. When the ring structure has been
once synthesized, further changes can take place either by the addition
of side-chains to the ring or by the condensation of two or more rings.
In this way the great multitude of aromatic products present in the
higher plants may arise.

Thus the cell can be pictured as a colloidal solution of proteins
endowed with the properties of matter in mass and surrounded by a
permeable cell-wall of cellulose. The colloidal solution contains liquid
and solid particles of very varied chemical composition. In the protoplasm
are spaces, vacuoles, filled with cell -sap also containing many and
various substances in solution. Throughout the protoplasm, which
probably has an organized structure, many kinds of chemical reactions,
are continually in progress, some being the converse of others, as for
instance those of oxidation and reduction which can take place side by
side in the same cell.

Next will be considered the chemical reactions by which the various
metabolic changes in the plant are brought about. How are these
processes controlled and how do they take place ?

There is a large group of organic substances, termed enzymes, many
of which are present in every plant. They have a certain characteristic in
common, i.e. they bring about chemical reactions in the plant without
undergoing any permanent change: in other words they are organic
catalysts. Many of these reactions, which take place in the cell at
ordinary temperatures with considerable rapidity, need prolonged heating
at high temperatures when brought about by artificial means. Enzymes

8 INTRODUCTION [CH.

can generally be extracted from the plant by water, especially if the
tissues are thoroughly disintegrated. Their chemical constitution is
at present unknown, and they are usually destroyed by temperatures
greater than 60°C. Moreover, many of the processes which they control
in the plant can be brought about by them in vitro under suitable
conditions, and it is by means of such experiments that information as
r to their rdle in plant metabolism has been ascertained. The majority of
known enzymes control both hydrolysis and its converse, synthesis by
condensation with elimination of water, but under artificial conditions
hydrolysis most frequently occurs. The enzyme, diastase, for instance,
found in all starch -containing plants hydrolyzes in vitro starch to dextrin
and maltose. Similarly the enzyme, maltase, hydrolyzes maltose into
glucose. Other enzymes hydrolyze proteins into amino-acids, and others,
again, hydrolyze fats into fatty acids and glycerol.

Until fairly recently the fact escaped notice that such reactions are
reversible, and that these enzymes in situ in the plant may, according
to the conditions, control not only the hydrolytic but also the cor-
responding synthetic process. The latter may also be brought about,
though not readily, in vitro. This, and other evidence, leads us to
believe that enzymes in the plant control the reactions in both directions.

Hydrolysis, and synthesis with elimination of water are not however
the only processes catalyzed by enzymes. There is another type of these
catalysts, the oxidizing enzymes, which bring about oxidation of sub-
$tances in the plant, notably of aromatics. In addition, there is the
enzyme, zymase, which decomposes sugar with the production of alcohol
and carbon dioxide.

The question which now arises is — How many reactions in the plant
are catalyzed by enzymes ? It is conceivable that a greater number of
enzymes may exist than are at present known, but that they are unable
to be extracted by our present methods of isolation. A certain number
of reactions probably take place in the cell-sap between the substances
in solution ; others are catalyzed by enzymes which are supposed to be
intimately connected with the protoplasm, but there are an enormous
number to which there is at present no clue as to how they are brought
about, such, for instance, as the synthesis of carbohydrates from carbon
dioxide and water, and the formation of the benzene ring from the open
carbon chain. Such processes are -usually said to be controlled by the
"living protoplasm," but what exactly is the significance of this
expression is at present beyond our knowledge.

Finally, also, little is known of the question as to how the various

i] INTRODUCTION 9

lines of metabolic syntheses in different parts of plants are regulated
and correlated with each othe.r. Some of the phenomena involved are
shortly outlined as follows. There is undoubtedly, under suitable
conditions, a constant synthesis of sugars in the leave's. In all pro-
bability aromatic substances are also synthesized in the same organs,
for there is evidence that there is an increase of these compounds in the
leaf if translocation through the petiole is prevented. It is possible that
^amino-acids also are formed in the leaf. The above products are
constantly translocated to the growing organs as material for growth.
They may, nevertheless, be temporarily stored in the tissues where they
have been synthesized, and of this there is evidence in at least one case,
e.g. starch in the leaf. But, apart from the immediate use for growth,
there is in practically every plant, some tissue where, owing to some
unknown stimulus (causing probably changes in permeability of the
cell-membranes), accumulation of compounds occurs. This accumulation
is characteristic of organs from which growth will take place when it is
impossible for the plant to obtain fresh supplies by carbon assimilation,
as, for example, of bulbs, rhizomes, tubers, buds, seeds, fruits and woody
tissues. In these cases, in due time, the products stored supply the
growing shoots.

During storage, simple sugars, amino-acids, etc. have been condensed
to form insoluble, colloidal, or large molecules of starch, fats, aleurone,
cane-sugar, etc. These will remain until they are hydrolyzed by enzymes
when they can supply the growing shoots. Such stores are termed
"reserve materials." The actual stimuli involved in bringing about and
regulating this storage are unknown, but they are probably connected
with the life cycle of the particular plant under consideration and its
adaptation to external conditions.

REFERENCES

1. Abderhalden, B. Biochemisches Handlexikon. Berlin, 1911.

2. Allen's Commercial Organic Analysis. London, 1909-1917.

3. Cole, S. W. Practical Physiological Chemistry. Cambridge, 1919. 5th ed.

4. Czapek, P. Biochemie der Pflanzen. Jena, 1905.

5. Haas, P., and Hill, T. G. The Chemistry of Plant Products. London,
1917. 2nd ed.

6. Palladin, V. I. Plant Physiology. Edited by B. E. Livingston. Philadelphia,
1918.

7. Plimmer, R. H. A. Practical Organic and Biochemistry. London, 1918.
3rd ed.

CHAPTEE II

THE COLLOIDAL STATE

MANY of the substances of which the plant is built up exist in the living
cell in the colloidal state, and it is therefore important that some account
should be given of this condition of matter.

There are many organic products found in the plant (and also in the
animal), such as starch, various proteins, gums, etc., that apparently dis-
solve in water, giving a solution which, as a rule, only differs from an
ordinary solution by being opalescent. In addition, it has been known
for a long time that various inorganic substances, such as sulphides of
arsenic and antimony, hydroxide of iron, and also certain metals (gold,
silver), can, by special methods, be obtained in "solution," though in
ordinary circumstances they are quite insoluble. The above examples
are representative of colloidal solutions.

A property which all the above solutions possess is that the substance
dissolved will not pass through a parchment membrane, i.e. will not
dialyze, whereas if a solution of sodium chloride in water is separated
from pure water by a parchment membrane, the salt will pass through
the membrane until the concentration of the sodium chloride is equal
on either side of it.

The conclusion drawn from investigations of various kinds is that in
the colloidal solutions the substances dissolved exist in the state, either
of aggregates of molecules, or of very large molecules, and hence are
unable to pass through the pores of the parchment.

Moreover, certain distinctions can be drawn between colloidal solu-
tions : some, like those of gold, silver, metallic sulphides, hydroxides and
in fact most inorganic substances, are very sensitive to the presence of
small amounts of inorganic salts, i.e. electrolytes, and are precipitated
by them, but will not as a rule go into solution again. Also such col-
loidal solutions are very little more viscous than pure water. The organic
substances, on the other hand, are only precipitated from colloidal solu-
tions by comparatively large quantities of electrolytes. The viscosity,
moreover, of these solutions is greater than that of water, and is, in fact,
considerable, even if the percentage of dissolved matter is small.

CH. n] THE COLLOIDAL STATE 11

Hence two terms have been employed for the above-mentioned
types of colloidal solutions: those of gold, silver, etc., are termed suspen-
soids (suspensoid sols) : those of starch, proteins, etc., emulsoids (emul-
soid sols).

The essential feature of both forms is that they are systems consisting
of two phases, or conditions of matter, known respectively as the " dis-
persed " phase and the " continuous " phase.

A suspensoid may be denned as having a dispersed phase composed
of ultramicroscopic particles or aggregates of molecules suspended in a
continuous phase composed of a liquid.

An emulsoid may be defined as having a dispersed phase composed
of ultramicroscopic drops of a highly concentrated solution of the sub-
stance suspended in a continuous phase composed of a dilute solution of
the same substance.

As a rule, therefore, the difference between a suspensoid and an
emulsoid is that, whereas in the former the liquid is restricted to the
continuous phase, and the solid to the dispersed phase, in an emulsoid
both phases are liquid, though containing different proportions of the
dissolved substance.

The terms suspensoid and emulsoid are used on account of the re-
semblance of these states of matter respectively to suspensions and
emulsions. If microscopic particles of a solid are shaken up in water,
what is known as a suspension is obtained ; in time, however, the solid
particles, if heavy enough, will settle and separate from the water, and
the whole process can be repeated. Thus a suspension differs from a
suspensoid solution in that the latter is stable, though, if precipitated,
the reaction is usually not reversible.

If two liquids which are insoluble in each other, such as oil and
water, are shaken up together, finely divided drops of oil in water are
obtained. This is known as an emulsion. In time, however, the oil
separates from the water, because the tension on the films of water
separating the oil drops, when in contact, is too great, and they break,
with the result that the oil drops coalesce. But if, instead of water, a
solution of soap, saponins, or certain other substances is used, the surface
tension of the water is so lowered that the films of soap solution separating
the oil drops are permanent, and a system is obtained consisting of minute
drops of oil separated by soap solution. This system resembles an organic
colloidal solution, as, for instance, that of protein in which we suppose
a concentrated solution of protein exists in drops separated by a dilute
solution of protein. Milk and latex constitute natural emulsions.

12 THE COLLOIDAL STATE [CH.

Expt. 1. Formation of a suspension. Precipitate a solution of barium chloride
with some sulphuric acid and shake up well the fine precipitate of barium sulphate.
Note the gradual settling of the precipitate.

Expt. 2. Formation of an emulsion. Take a drop of olive oil in a test-tube and
half fill the tube with alcohol. Shake well and pour into a beaker of water. A fine
white emulsion of oil in water will be formed from which the oil will not separate.
By this method the oil is obtained in such small drops that stability is ensured.

Take about equal quantities of olive oil in two test-tubes and add an equal
quantity of water to each. To one tube add a drop or two of 10°/0 caustic alkali
solution. Shake both test-tubes well. An emulsion is formed in both, but in the
tube without alkali the oil will separate out on standing. In the other tube the
emulsion is permanent. This is due to the fact that the olive oil (unless specially
purified) contains some free fatty acid. The latter forms soap with the alkali (see
p. 82) and renders the emulsion permanent.

Expt. 3. Preparation of suspensoid sols, (a) Gold. "Take two IGOc.c. measuring
cylinders and thoroughly clean them with nitric acid, and afterwards wash well with
freshly distilled water. In one make a 0'5°/ft solution of tannic acid (using the purest
sample obtainable) in water. In the other. 2 c.c. of commercial 1 % gold chloride are
made up to 100 c.c. with water. Use freshly distilled water in both cases. Mix equal
portions of the two solutions in a clean beaker. A purple colloidal solution of gold
will be formed. If three parts of the chloride solution are mixed with one part of the
tannin solution, and both solutions heated before mixing, a red colloidal solution is
obtained. (6) Silver. Take 5 c.c. of a 1 % solution of silver nitrate and add dilute am-
monia solution until the precipitate first formed just disappears, and then dilute with
100 c.c. of water. Mix equal volumes of this solution and the tannic acid prepared
for (a). A colloidal solution of silver will be formed which is clear brown by trans-
mitted light, but has a green fluorescence by reflected light, (c) Ferric hydroxide.
Take 5 c.c. of a filtered 33 % solution of ferric chloride and pour into 500 c.c. of
boiling distilled water in a beaker. A colloidal ferric hydroxide sol is formed and the
colour changes to a deep brown-red. The yellow solution of ferric chloride is de-
composed by excess of water with the production of a soluble colloidal form of ferric
hydroxide, and hydrochloric acid is set free, (d) Arsenic trisulpjiide. Take 2 grns.
of arsenious acid and boil with 150 c.c. of distilled water, filter and cool. Then pass
sulphuretted hydrogen through the solution. A colloidal solution of the sulphide is
formed which is orange, with a greenish surface.

The above sols should be kept for further experiment [see Expt. 8J

Expt. 4. Preparation of emulsoid sols, (a) Starch. Weigh out 2 gms. of dry starch,
and mix well with a little cold distilled water. Boil 100 c.c. of distilled water in a
flask, and, when boiling, pour in the starch paste and boil for a few minutes longer,
stirring well all the time. A colloidal solution of starch is obtained which is faintly
opalescent. It is not affected by heating and does not change its state on cooling.
(6) Gum arabic. Make a 5 % solution of gum arabic by boiling 5 gms. with 100 c.c.
of distilled water. Note that a sticky or viscous solution is formed which froths on
shaking, (c) Protein. Weigh out 10 gms. of white flour and add 100 c.c. of distilled
water. Let the mixture stand for 2 or 3 hours and then filter. The extract contains
protein. Note that the solution froths on shaking, (d) Soap. Make a 5-10% solution

n] THE COLLOIDAL STATE 13

of soap in distilled water. It is opalescent and froths strongly, (e) Chlorophyll.
[See Expt. 32.]

The above sols should be kept for further experiment [see Expt. 9].

Expt. 5. Dialysis of starch and salt solution. Make a 2 % solution of starch in
water, as in Expt. 2, and mix it with an equal volume of a 2 °/0 solution of sodium
chloride in water. Pour the mixture into a parchment dialyzer and dialyze in a
beaker of distilled water. (The dialyzer should first be carefully tested to ascertain
if there be a leak.) Test the liquid in the beaker with solutions of both iodine and
silver nitrate. Some precipitate of chloride will be given, but no blue colour with
iodine. After 24 hours, test the liquid again. There will be an increased amount of
silver chloride formed, but a negative result with iodine. On addition of iodine to
the liquid in the dialyzer, a blue colour is obtained. Hence we may assume that the
colloidal starch does not pass through the membrane.

Some substances, such as gelatine (animal) and agar (vegetable), are
only in the emulsoid condition at a raised temperature. When cold
they set to form a " gel," in which the particles of the dispersed phase are
no longer separate but united to make a solid. Silicic acid, the best known
inorganic emulsoid, also sets to a gel on standing, either spontaneously
or on addition of electrolytes. It is of classical interest since it was the
substance largely used by Graham, the first worker on colloids.

Expt. 6. Preparation of gels, (a) Agar. Weigh out 2 gms. of agar and put it
to soak in 100 c.c. of distilled water for an hour or two. Then boil : the agar gives a
thick opalescent solution (sol) which sets to a gel on cooling. On warming, the gel
again becomes a sol, and, on cooling, again sets to a gel. Thus the change is a
reversible one. Agar is a mucilage which is obtained from certain genera of the
Rhodophyceae (see p. 49). (6) Silicic acid. Weigh out 20 gms. of commercial "water-
glass" syrup (a concentrated solution of sodium silicate) and dilute with 100 c.c. of
freshly boiled distilled water (free from carbon dioxide). Pour 75 c.c. of this solution
into a mixture of 25 c.c. of concentrated hydrochloric acid and 75 c.c. of water.
Dialyze the mixture in a parchment dialyzer against running water for 3-4 hours.
If to the dialyzed liquid a little very dilute ammonia is added, a gel will be formed
in the course of a few hours. In this case, however, the process is irreversible, that
is the gel cannot be reconverted again into the sol.

An interesting point in connexion with the colloidal state is that
emphasized by Ostwald, i.e. that this condition is a state, not a type,
of matter. Further, substances in the colloidal state do not constitute
a definite class. It is reasonable to suppose that all substances which
exist in the colloidal state can, under suitable conditions, also exist in
the crystalline state, and vice versa. Further, the continuous phase is
not always water. Sodium chloride, which is a very definite crystalloid,
can be obtained in the colloidal state in petroleum ether. Most metals,
even the alkali metals, have been obtained in colloidal solution : also a
great many metallic oxides, hydroxides and sulphides.

14 THE COLLOIDAL STATE [CH.

The colloidal phases so far dealt with can be tabulated as follows1:
disperse continuous

liquid solid gels

solid liquid suspensoids

liquid liquid emulsoids

Some of the properties of colloidal solutions may now be considered.
Appoint that has already been emphasized in the previous chapter is
that the surface of particles in the colloidal state is very great in pro-
portion to their mass. Such particles, moreover, unlike ions and small
molecules in true solution, possess the properties of the surfaces of matter
in mass, as, for instance, those connected with surface tension, electrical
charge, etc., and these are especially marked on account of the propor-
tionately large surfaces involved. Other properties are their inability,
as a rule, to exert an osmotic pressure, to raise the boiling point, and to
lower the freezing point of water. Some of the metallic suspensoids are
characterized by their colour, this being red, purple or blue as in the case
of gold sols.

An apparatus, by means of which the colloidal state can be demon-
strated ocularly, is the ultramicroscope. iThis is a special form of micro-
scope in which a powerful beam of light is directed upon a colloidal solution,

j which is then seen to contain a number of particles in rapid motion.

""When analyzed by special methods, this motion has been found to be
identical with that shown by much larger, though still microscopic,
particles, which has been termed Brownian movement.

Expt. 7. Demonstration of Brownian movement of microscopic particles. Mount a
little gamboge in water and examine under the high power of a microscope. The
particles will be seen to be in rapid motion.

It has been shown that Brownian movement is the outcome of the
movement of the molecules of the liquid in which the particles are
suspended. This movement is one of the factors which keeps the sol
stable and prevents the particles from "settling" as in the case of a
true suspension.

Another factor tending to keep the sol stable is the electrical charge
borne by the particles. It is commonly known that there is usually a
difference of potential between the contact surfaces of phases. If the

1 There are also the following combinations (Bayliss, 1) :
disperse continuous

gas liquid foam

liquid gas fog

solid gas tobacco smoke

solid solid ruby glass (colloidal sol of gold in glass).

n] THE COLLOIDAL STATE 15

particles in a colloidal solution all have the same charge, then they will
tend to repulse one another mutually. It is found that the particles
are charged, but the origin of the charge is not always clear. Sometimes
if the substance in colloidal state is capable of electrolytic dissociation,
the charge may arise in this way. Substances, however, as already men-
tioned, which are not dissociated may also bear a charge, and most
frequently it is a negative one. It follows, then, that when an electro-
lyte is added to a colloidal solution, the charges on the colloidal particles
are neutralized by the oppositely charged ions of the electrolyte, and
they coalesce together and are precipitated.

As regards their behaviour to electrolytes the two classes, suspen-
soids and emulsoids, are very different. The suspensoids are very sensi-
tive to traces of electrolytes, and, as they usually have a negative charge,
it is the cation of the electrolyte which is the active ion; and of such,
less of a divalent ion, than of a monovalent ion, is needed for precipita-
tion and still less of a trivalent ion.

The emulsoids are far less sensitive to electrolytes in solution than
the suspensoids ; in fact, electrolytes, such as neutral alkali salts, must
be added in very large quantities to emulsoids before precipitation takes
place. Also, as a rule, whereas the precipitation of suspensoids is irre-
versible, that of emulsoids is reversible, that is, they pass into solution
again on addition of water. In the case of an emulsoid in neutral solu-
tion this form of precipitation, unlike that of the suspensoids, may be
regarded as consisting of two processes. First, a process analogous to
that of " salting out " of soaps, esters, etc., in organic chemistry, which
is, in effect, a withdrawal of water from one phase into another. Secondly,
the precipitation is also affected to some extent by the valency of the
ions of the salt used in precipitation.

When, however, a neutral solution of such an emulsoid as protein is
made either slightly acid or alkaline, its behaviour towards neutral salts
becomes altered. The precipitating power of salts in acid or alkaline
medium is now in accordance with that on suspensoids. In alkaline
solution the coagulating power of a salt depends on the valency of the
cation; in an acid solution it depends on the valency of the anion.

The behaviour of proteins in acid and alkaline media is undoubtedly
due to the fact that they are built up of amino-acids containing both
ammo and carboxyl groups. Such molecules may behave either as an
acid or a base with the formation of salts. These are subject to electro-
lytic dissociation and hence acquire an electric charge. Such substances
have been termed "amphoteric electrolytes" (see p. 120).

16 THE COLLOIDAL STATE [CH. n

Expt. 8. Precipitation of suspensoid sols by electrolytes. The sols of gold, .silver
and arsenious sulphide carry an electro-negative charge : hence they are most readily
precipitated by di- or tri-valent positive ions, such as Ba" or Al"'. Add a few drops
of barium chloride solution to the three sols (Expt. 3) respectively, and note that
they are precipitated, though some time may elapse before the precipitation is
complete. The ferric hydroxide sol, on the contrary, carries a positive charge. Hence
it is most readily precipitated by a sulphate or phosphate. If a drop of sodium
sulphate solution is added while the sol is hot, it is immediately precipitated.

Expt. 9. Precipitation of emulsoid sols by electrolytes. Saturate the starch, protein
and soap solutions prepared in Expt. 4 with solid ammonium sulphate. Precipitation
takes place in each case, and it is seen how large a quantity of electrolyte is needed
for the "salting out" of emulsoid sols. Filter off the protein precipitate and suspend
in distilled water. It will go into solution again, showing that the reaction is
reversible.

REFERENCES

1. Bayliss, W. M. Principles of General Physiology. London, 1918. 2nd ed.

2. Burton, B. F. The Physical Properties of Colloidal Solutions. London,
1916.

3. Hatschek, B. An Introduction to the Physics and Chemistry of Colloids.
London, 1919. 3rd ed.

4. Philip, J. C. Physical Chemistry : its Bearing on Biology and Medicine.
London, 1913. 2nd ed.

5. Taylor, W. W. The Chemistry of Colloids. London, 1915.

CHAPTER III

ENZYME ACTION

SOME indication has been given in the previous chapter of the large
number of complex processes which take place in the plant, and it has
been mentioned that many of these aye controlled by enzymes.

The most remarkable feature in connexion with the chemical pro-
cesses of plant metabolism is the ease and rapidity with which, at
ordinary temperatures, chemical reactions take place, when under artificial
conditions they need a much longer time and higher temperatures.

It has been found that many of the chemical reactions in the plant
can be brought about in vitro on addition of certain substances which
can be extracted from the plant. These substances are known as enzymes.
It is the property of enzymes that they are able to accelerate reactions
which, in their absence, would only take place very slowly. The enzyme
cannot initiate these reactions and does not form part of their final
products.

Some inorganic substances have the same property of accelerating
reactions, and such substances are termed catalysts. For example, when
water is added to ethyl acetate, the latter begins to be decomposed slowly
into ethyl alcohol and acetic acid :

ethyl acetate -I- water — >• ethyl alcohol + acetic acid,

but if, in addition, some hydrochloric acid is added, hydrolysis takes place
with much greater rapidity, and at the end of the reaction the hydrochloric
acid is found unchanged. Hence in this case hydrochloric acid is an
inorganic catalyst. Many other similar instances are known as, for
example, the catalyzing effect of a small quantity of manganese
dioxide which brings about the liberation of oxygen from potassium
chlorate at a much lower temperature than by heat alone.

By analogy, therefore, an enzyme may be defined as an organic
catalyst produced by the plant.

Another point in connexion with the above-mentioned reaction of
water with ethyl acetate, is the fact of its being representative of the
type known as reversible. After a certain amount of acetic acid and
ethyl alcohol has been formed, these recombine to form ethyl acetate
until in time a certain point of equilibrium is reached. Since the same
o. 2

18 . ENZYME ACTION [CH.

point of equilibrium is reached whether hydrochloric acid is used or not,
it is obvious that the hydrochloric acid accelerates the reaction in both
directions :

ethyl acetate 4- water ^±: ethyl alcohol -I- acetic acid.

Such a reaction is termed a reversible one. Many of the processes
accelerated by enzymes in the plant are reversible, and there is reason
to believe that the enzyme accelerates the reaction in both directions.

The substance upon which the enzyme acts is termed the substrate,
and it is supposed that some kind of loose combination occurs between
these two substances. The enzyme is unaltered when the reaction is
complete, unless it is affected by the products formed.

The enzymes are very widely distributed and form constituents of
all living cells, though all tissues do not necessarily contain the same
enzymes.

There is no doubt that many enzymes are specific, in which case
an enzyme can only accelerate one reaction, or one class of reaction.
We cannot be sure that any enzyme is specific and different from all
others, until it has been proved that it accelerates one process which is
incapable of being accelerated by any other enzyme. It is possible
that some enzymes, to which separate names have been given, are really
identical.

Most of the plant enzymes are soluble in water and dilute glycerol
and sometimes in dilute alcohol. Some can be extracted by simply
macerating the tissues with water; others are more intimately connected
with the protoplasm, and are only extracted if the protoplasm is killed
by certain reagents, of which those most frequently employed are toluol
and chloroform. These substances kill the protoplasm and do not, in
many cases, affect the enzyme. After the death of the protoplasm, the
enzymes are more readily extracted from the cell. From aqueous
solutions enzymes can usually be precipitated by adding strong alcohol.

The majority of enzymes are destroyed by raising the temperature
above 60° C. In vitro their reactions are generally carried out most
rapidly between the temperatures of 35-45° C.

In performing experiments with enzymes in vitro, it is always
necessary to add an antiseptic, otherwise the reaction to be studied will
be masked or entirely superseded by the action of bacteria unavoidably
present. Toluol and chloroform mentioned above, as well as thymol,
may be used. These reagents prevent any bacterial action from taking
place. Some enzymes, however, are susceptible to chloroform, as, for
instance, maltase.

in] ENZYME ACTION 19

The chemical nature of enzymes is at present unknown, because it
is difficult to purify them without destroying them, and hence to obtain
them of sufficient purity for chemical analysis. They were originally
thought to be proteins, but with the improvements in methods for
purification, it has been found that the protein reactions disappear,
although the enzyme activity does not decrease. In solution they exist
in the colloidal condition.

The questions as to their origin and their relation to the protoplasm
cannot yet be answered with any certainty. It is also impossible to say
whether the majority of chemical processes in the plant are catalyzed by
enzymes.

A feature of enzyme action which is of considerable interest and
which has already been mentioned is the question as to whether enzymes
catalyze the synthetic as well as the hydrolytic reaction. There is
evidence that this is so, since, in many cases, the hydrolysis is not
complete. If the enzyme were a catalyst in one direction only, the
reaction would be complete. Further evidence is supplied by the fact
that, under suitable conditions, i.e. strong concentration of the substances
from which synthesis is to take place, certain syntheses have been carried
out in vitro. As an example may be quoted the synthesis of maltose
from a concentrated solution of glucose by maltase (Bayliss, 2).

In the living cell it is supposed that the hydrolysis and synthesis
are balanced. On the " death " of the protoplasm, which may be caused
by mechanical injury, vapour of chloroform or toluol, etc. (Armstrong, 7, 8),
the reactions catalyzed by enzymes cease to be balanced and proceed almost
always in the direction of hydrolysis and the splitting up of more complex
into simpler substances. This phenomenon is obvious when any of the
products can be recognized by smell or colour, as, for instance, the smell
of benzaldehyde on injuring leaves of plants containing cyanogen etic
glucosides (see p. 146), or the production of coloured oxidation products
when some of the aromatic glucosides are decomposed (see p. 113).

If plant tissuesare disintegrated, and the mass is kept at a temperature
of about 38° C., the above-mentioned hydrolytic processes continue to be
catalyzed by the enzymes present until equilibrium is reached, which will
be near complete hydrolysis, especially if water has been added. Such
a process is termed "autolysis."

The chief plant enzymes may be classified under the following
headings :

A. Hydrolytic enzymes. These constitute by far the greater number
of known enzymes. In their activity they either add or eliminate water.

2—2

20 ENZYME ACTION [CH.

According to the substances upon which they act (the substrates)
they may be further sub-classified as follows :

(1) Fat-splitting enzymes, which hydrolyze fats into fatty acids
and glycerol : lipase.

(2) Carbohydrate-splitting enzymes :

Diastase, which hydrolyzes starch into dextrin and maltose.

Invertase, which hydrolyzes cane-sugar into dextrose and laevulose.

^laltase, which hydrolyzes maltose into dextrose.

Inulase, which hydrolyzes inulin into laevulose.

Cytase, which hydrolyzes hernicellulose into mannose and galactose.

Pectinase, which hydrolyzes pectic compounds into arabinose.

(3) Glucoside-splitting enzymes :

Emulsin, which hydrolyzes amygdalin into benzaldehyde, prussic
acid and glucose.

Myrosin, which hydrolyzes sinigrin into allylisothiocyanate, potassium
hydrogen sulphate and glucose.

(4) Protein-splitting enzymes (proteases) :

Pepsin-like enzymes, which hydrolyze proteins into albumoses and
peptones.

Erepsin-like enzymes, which hydrolyze peptones into polypeptides
and amino-acids.

B. Oxidizing enzymes :

Peroxidases, which decompose peroxides and set free oxygen in
the active state, probably as atomic oxygen.

Catalases, which decompose hydrogen peroxide and set free oxygen
in the molecular condition.

C. Fermenting enzymes: Zymase of yeast and also of higher plants,
which brings about the decomposition of certain hexoses, such as glucose,
with the formation of alcohol and carbon dioxide. Probably a series of
reactions including oxidation, reduction and hydrolysis are involved.

D. Coagulating enzymes : Pectase, which coagulates soluble pectic
compounds.

E. Reducing enzymes : Reductase. The precise function of these
enzymes is unknown.

Most of these various classes of enzymes will be dealt with in detail
in connexion with the chemical substances on which they react.

An excellent demonstration of the fact that a single cell may contain
all the various enzymes connected with the processes of metabolism is

in] ENZYME ACTION 21

afforded by the unicellular Fungus, Yeast (Saccharomyces), of which many
species and varieties are known. The feature of special interest in
connexion with the Yeast plant is its power of fermenting hexoses, with
the formation of alcohol and carbon dioxide, the process being carried
out by means of an enzyme, zymase. The reaction is generally
represented as follows, though there is little doubt that several stages
are involved :

In addition to zymase, yeast contains the enzymes, invertase, protease,
peroxidase, catalase, reductase, glycogenase, carboxylase, a glucoside-
splitting enzyme, and some form of diastatic enzyme. The carboxylase
decomposes a large number of keto-acids, of which the most impor-
tant is pyruvic acid CH3 • CO • COOH. The reaction involves the
formation of the corresponding aldehyde with the evolution of carbon
dioxide. Yeast also stores, as a reserve material, the polysaccharide,
glycogen, which occurs in animal tissues though it is rarely found in
plants : this is hydrolyzed by glycogenase into a monosaccharide. Finally,
yeast contains invertase, and most species, in addition, maltase, but from
a few species the latter enzyme is absent. Hence yeasts are able to
ferment the disaccharides, cane-sugar and maltose, since they can first
hydrolyze them to monosaccharides.

As in the case of the enzymes of other tissues, those of yeast can be
made to carry out their functions after the death of the living protoplasm.
The method of demonstrating this is to "kill" the cells by means of
drying at 25-30° C., by treatment with a mixture of alcohol and ether,
or by treatment with acetone and ether. In this way the protoplasm is
destroyed, but the enzymes remain uninjured. Yeast treated thus has
been termed " zymin."

From zymin some of the enzymes, e.g. invertase and the glucoside-
splitting enzyme, can be extracted with water: other enzymes, e.g. zymase
and maltase, are not so readily extracted. From the living cells the
enzymes are only obtained with difficulty, the extraction of yeast juice,
containing zymase and other enzymes, needing, by Buchner's method,
a pressure as great as 500 atmospheres.

In connexion with alcoholic fermentation by zymase, the following
point is of special interest. For carrying out this process, two other
substances are necessary in addition to the enzyme, i.e. a co-enzyme of
unknown nature and a phosphate. The separation of zymase from the
co-enzyme has been accomplished by filtering expressed (Buchner) yeast
juice through a special form of gelatine filter under a pressure of 50

22 ENZYME ACTION [CH.

atmospheres. The phosphate and co-enzyme can also be removed from
zymin by washing with water. The washed residue is then found to be
incapable of fermentation, as also are the washings. If, however, the
boiled washings are added to the washed residue, the system is synthesized
and can now carry out fermentation again. The chemical nature of the
co-enzyme, which is thermostable, and the precise part played by it in
the process, are as yet unknown (Harden, 4).

Expt. 10. Preparation of zymin. (a) By alcohol and ether. For the following
experiments fresh yeast should be used which has been washed several times with
distilled water and dried on a filter-pump. Weigh out 25 gms. of yeast and stir
it up well in a mixture of 300 c.c. of absolute alcohol and 100 c.c. of ether for 4-5
minutes, and then filter on a filter-pump. Next wash the yeast with 50 c.c. of
ether. Then spread it out on a piece of filter-paper and allow it to dry for one hour.
(6) By acetone and ether. Take 50 gms. of yeast and stir it into 300 c.c. of acetone.
Continue stirring for 10 minutes, and filter on a filter-pump. The mass is then
mixed with 100 c.c. of acetone for 2 minutes and again filtered. The residue is
roughly powdered, well-kneaded with 25 c.c. of ether for 3 minutes, filtered, drained
and spread on filter-paper for an hour in the air. It can be finally dried at 45° C. for
24 hours.

Expt. 11. Action of zymase. Fill a small test-tube with 40% glucose solution.
Add 2 gms. of the zymin (from Expt. 10) for each 10 c.c. of solution and a little
toluol, and shake. Then fit a slightly larger test-tube over the mouth of the first
tube. Now invert. A small quantity of air will rise to the top of the first tube
which, however, will remain filled with the sugar solution. Place the tubes either in
an incubator or water-bath at 22-25° C. A control set of tubes should also be arranged
containing sugar solution and zymin which has been previously well boiled in a little
water. Bubbles of carbon dioxide will be evolved from the unboiled zymin.

Expt. 12. Action of maltase. (Harden and Zilva, 12.) Into two test-tubes, (a) and
(6), put the following :

(a) 20 c.c. of a 2 % solution of maltose -f 0*5 gm. of zymin.

(6) 20 c.c. of the same solution of maltose + 0'5 grn. of zymin which has been
well boiled in distilled water.

Plug both tubes with cotton -wool, after adding a few drops of toluol, and place
in an incubator at 38° C. for 12-24 hrs. Test the liquid in both tubes by making the
osazone (see p. 49). Glucosazone will crystallize out in tube (a), maltosazone in
tube (6).

Expt. 13. Action of peroxidase. (Harden and Zilva, 12.) Into four small
evaporating dishes, (a), (6), (c] and (d), put the following :

(a) A suspension of 0'5 gm. of fresh yeast in 10 c.c. distilled water + 1 c.c. of
benzidine solution (1 °/0 in 50% alcohol) + 2-3 drops of hydrogen peroxide (20 vols.).

(b) A suspension of 0'5 gm. of zymin in 10 c.c. of distilled water + 1 c.c. of
benzidine solution + 2-3 drops of hydrogen peroxide.

(c) A suspension of 0'5 gm. of washed zymin in 10 c.c. of distilled water + 1 c.c.

in] ENZYME ACTION 23

of benzidine solution + 2-3 drops of hydrogen peroxide. (The zymin is washed by
putting it on a double folded filter-paper in a funnel and adding distilled water from
time to time. 50 c.c. of water should be used for 0'5 gin. of zymin.)

(d) A suspension of 0'5 gm. of washed zymin in 10 c.c. of washings + 1 c.c. of
benzidine solution + 2-3 drops of hydrogen peroxide.

A blue colour will develop in (a) showing that fresh yeast contains a peroxidase
(see p. 109). A blue colour will also develop in (c) but not in (6) and (d). This is
explained by assuming that the zymin contains an inhibitor, not present in fresh
yeast, but which is developed during the preparation of the zymin, and that this
inhibitor can be washed away by water. On adding the washings to the washed
zymin the reaction is inhibited again.

Expt. 14. Action of catalase. (Harden and Zilva, 12.) Completely fill a test-tube
with hydrogen peroxide (20 vols.) solution which has been diluted with an equal
volume of water and add O'5-l gm. of zymin. Place the thumb firmly over the
mouth of the tube, invert and place the mouth under water in a small basin, clamp-
ing the tube in position. A rapid evolution of oxygen takes place. When the tube
is about three-fourths full of gas, close the mouth with the thumb while still under
water and remove the tube. Plunge a glowing splint into the gas and it will re-kindle
to a flame.

Expt. 15. Action of protease. Weigh out 10 gms. of white flour, and allow it to
extract with 100 c.c. of distilled water for one hour, shaking from time to time.
Then filter on a filter-pump. The extract will contain the "albumin, leucosin
(see p. 124). Into small flasks (a) and (b) put the following :

(d) 40 c.c. of the flour extract + 1 gm. of zymin + 1 c.c. of toluoL

(6) 40 c.c. of flour extract -f 1 gm. of boiled zymin -I- 1 c.«. of toluol.

Shake both tubes, plug with cotton-wool and place them in an incubator at 38° C.
for 48 hrs. After incubation, boil the liquid in both tubes, in order to coagulate
unaltered protein, and filter. To the filtrates of the respective tubes add bromine
water drop by drop (see p. 138). A pink, or purplish-pink colour, due to the presence
of tryptophane, will be formed in tube (a). Hence hydrolysis of protein has taken
place. Tube (6) will show no colour or only that due to bromine. Add a little amyl
alcohol to both tubes and shake. The alcohol will be coloured pink or purplish in
the tube giving the tryptophane reaction.

Expt. 16. Action of reductase. (Harden and Xorris, 11.) Take two small flasks,
(a) and (6), provided with well-fitting corks and put in the following:

(a) 1 gm. of zymin + 20 c.c. of distilled water + 0'5 c.c. of methylene blue
solution (made by diluting 5 c.c. of a saturated alcoholic solution to 200 c.c. with
distilled water).

(6) 1 gm. of boiled zymin 4- 20 c.c. of distilled water + 0'5 c.c. of methylene blue
solution.

Cork both tubes after adding a few drops of toluol and place in an incubator at
38° C. for 3 hours. The blue colour will practically disappear from tube (a) but will
remain in tube (6).

The methylene blue is reduced to a colourless leuco-compound which will become
blue again on re-oxidation.

24 ENZYME ACTION [CH.

Expt. 17. Enzyme actions of an aqueous extract of zymin. Weigh out 2 gms. of
zymin and place them on a double folded filter-paper in a funnel and wash with
80 c.c. of distilled water. With the filtrate make the following experiments.

(A) Action ofinvertase. (Harden and Zilva, 12.) Into two small flasks (a) and (6)
put the following :

(a) 10 c.c. of a 2 % solution of pure cane-sugar + 10 c.c. of the filtrate from
zymin.

(b) 10 c.c. of the same solution of cane-sugar + 10 c.c. of the boiled filtrate from
zymin.

Put both flasks in an incubator at 38° 0. After 30 minutes add equal quantities
(about 1-2 c.c.) of Fehling's solution to both test-tubes and boil (see p. 52). Tube (a)
will show considerable reduction of the Fehling. Tube (6) will show comparatively
little reduction, that which does take place probably being due to the sugar formed
by the action of glycogenase on stored glycogen.

(B) Action of the glucoside-splitting enzyme. (Caldwell and Courtauld, 9 ; Henry
and Auld, 13.) This enzyme will act upon the glucoside, amygdalin, which is present
in bitter almonds, with the production of glucose, benzaldehyde and prussic acid
(see p. 145). Into two small flasks (a) and (6) put the following :

(a) 20 c.c. of a 2 % solution of amygdalin + 20 c.c. of the filtrate from zymin.

(b) 20 c.c. of the same solution of amygdalin + 20 c.c. of the boiled filtrate from
zymin.

Add a few drops of toluol to both flasks and then cork, inserting, with the cork,
a strip of paper which has been dipped in solutions of picric acid and sodium
carbonate (see p. 146). Put both flasks in an incubator at 38° C. for 12-24 hours. The
picrate paper in flask («) will have reddened. Add a little Fehling's solution to the
liquid in the same flask and boil. The Fehling will be reduced. The liquid in flask
(b) will only reduce Fehling slightly [see Expt. A (6)] and the picrate paper will not
be reddened.

REFERENCES
BOOKS

1. Abderhalden, B. Biochemisches Handlexikou, v. Berlin, 1911.

2. Bayliss, W. M. The Nature of Enzyme Action. London, 1919. 4th ed.

3. Euler, H. General Chemistry of the Enzymes. Translated by T. H. Pope.
New York and London, 1912.

4. Harden, A. Alcoholic Fermentation. London, 1914. 2nd ed.

5. Vernon, H. M. Intracellular Enzymes. London, 1908.

6. Wohlgemuth, J. Grundriss der Fermentmethoden. Berlin, 1913.

PAPERS

7. Armstrong, H. B., and Armstrong, E. P. The Origin of Osmotic Effects.
III. The Function of Hormones in Stimulating Enzymic Change in Relation to
Narcosis and the Phenomena of Degenerative and Regenerative Change in Living
Structures. Proc. R. Soc., 1910, B Vol. 82, pp. 588-602. Ibid. IV. Note on the
Differential Septa in Plants with reference to the Translocation of Nutritive Materials.
Proc. R. Soc., 1912, B Vol. 84, pp. 226-229.

in] ENZYME ACTION 25

8. Armstrong, H. B., and Armstrong, B. P. The Function of Hormones
in regulating Metabolism. Ann. Bot., 1911, Vol. 25, pp. 507-519.

9. Caldwell, R. J., and Courtauld, S. L. Studies on Enzyme Action.
IX. The Enzymes of Yeast : Amygdalase. Proc. R. Soc., 1907, B Vol. 79,
pp. 350-359.

10. Harden, A. The Enzymes of Washed Zymin and Dried Yeast. I. Car-
boxylase. Biochem. J., 1913, Vol. 7, pp. ^214-217.

11. Harden, A., and Norris, R. V. The Enzymes of Washed Zymin and
Dried Yeast. II. Reductase. Biochem. «/., 1914, Vol. 8, pp. 100-106.

12. Harden, A., and Zilva, S. S. The Enzymes of Washed Zymin and Dried
Yeast. III. Peroxydase, Catalase, Invertase and Maltese. Biochem. J., 1914, Vol. 8,
pp. 217-226.

13. Henry, T. A., and Auld, S. J. M. On the Probable Existence of Emulsin
in Yeast. Proc. R. Soc., 1905, B Vol. 76, pp. 568-580.

CHAPTER IV

CARBON ASSIMILATION

THE fact has already been emphasized that the plant synthesizes all the
complex organic substances of which it is built from the simple com-
pounds, carbon dioxide, water and inorganic salts. The initial metabolic
process and the one from which all others have their starting-point is
that of a synthesis of a carbohydrate from carbon dioxide and water.
This synthesis can only be carried out in the light, and only in a green
plant, i.e. a plant containing chlorophyll. Chlorophyll may almost be con-
sidered the chemical substance of primary importance in the organic world,
for upon it depends the life of all plants and animals. Animals depend
for their existence on certain complex amino-acids, some of which they
are unable to synthesize for themselves, and which they derive from
plants. Plants in turn are unable to exist except by virtue of the pro-
perties of chlorophyll.

The property of chlorophyll which is so important is the power it
possesses of absorbing the radiant energy of the sun's rays and converting
it into chemical energy by means of which a carbohydrate is synthesized.
This summarizes the whole process, which, however, can scarcely be very
simple, and probably consists of several reactions at present undifferen-
tiated. If the formula for carbonic acid is compared with that of a simple
carbohydrate such as a tetrose, pentose or hexose, the following relation-
ship is seen :

H2CO3-^(H2CO)^ where x=4, 5 or 6,

that is, in the synthesis of a carbohydrate a reducing reaction must take
place.

Many hypotheses have been formulated as to the nature of these re-
actions. The one which has most frequently been advanced suggests
that formaldehyde is the first product of the synthesis from carbon
dioxide and water which takes place in the green plant ; that the re-
action involves reduction with elimination of oxygen :

H2CO3=H2CO + O2,
and that this product is later condensed to form a hexose,

CH. iv] CARBON ASSIMILATION 27

As the concentration of sugar increases in the cell, further condensation
may take place to form starch :

^(C6H1206) = (C6H1005)a;+^ H20.

The facts in agreement with these views are : first, in most plants a
volume of oxygen is given off approximately equivalent to the volume
of carbon dioxide absorbed ; secondly, in some plants starch, in others
sugar, is known to be produced during photosynthesis. The detection
of formaldehyde, either in the plant or in certain systems containing
chlorophyll, as a proof of its formation during photosynthesis, has been
shown to be invalid (see p. 36) (Jorgensen and Kidd, 2).

The value and significance of this reducing reaction is seen when it
is realized that, by oxidation of the carbohydrates synthesized, energy is
produced to supply the needs of the whole metabolism of the plant
(see p. 5).

In the chemical treatment of the subject of carbon assimilation, some
of the chemical properties of chlorophyll will first be considered, and,
later, its behaviour under certain conditions : the chemistry, however, of
the phenomenon itself is as yet unknown.

The following account, as far as it concerns chlorophyll, and the
accompanying experiments are taken from a resume (Jorgensen and
Stiles, 3) of the original w,ork (Willstatter und Stoll, 1) upon which the
entire knowledge of the subject is based.

CHLOROPHYLL.

Our knowledge of the chemistry of chlorophyll has, within recent
years, been set upon a firm experimental basis (Willstatter und Stoll, 1).
The results which have been arrived at may broadly be summarized as
follows :

In all plants examined the chloroplastids contain four pigments, of
which two (termed respectively chlorophylls a and b) are green, and
two are yellow. They occur in about the following proportions in fresh
leaves :

(Chlorophyll a ... C55H72O5N4Mg . . . 2 pts per 1000
{Chlorophyll b ... C55H7006N4Mg ... | „

Yellow iCar°tin ••• C4°H56 * »

IXanthophyll ...CJBLp, J „

A point of great interest in connexion with chlorophyll is thatTit
contains magnesium to the extent of 2'7 % but no other metal is present.
Chlorophyll a, when isolated, is a blue-black solid giving a green-blue

28 CARBON ASSIMILATION [CH.

solution in the solvents in which it is soluble, i.e. ethyl alcohol, acetone,
chloroform, ether, carbon bisulphide, pyridine and benzene. Chlorophyll
b, when isolated, is a green-black solid giving a pure green solution : it
has much the same solubilities as chlorophyll a. The two chlorophylls,
however, can be separated by their different solubilities in methyl
alcohol. Both can be obtained in microscopic crystals.

Carotin crystallizes in orange-red crystals, and xanthophyll in yellow
crystals.

In the chloroplastids these pigments occur mixed with various colour-
less substances, fats, waxes, and salts of fatty acids.

When chlorophyll is spoken of, it will be understood to refer to the
green pigments and not to the yellow.

The pure pigments, when isolated, are readily soluble in acetone, ether
and benzene. When very thoroughly dried nettle leaves are treated
with pure acetone, no green colour is extracted, but if a few drops of
water are added, the extract becomes green. Also if acetone is poured
on to fresh leaves, the pigment is extracted. The explanation offered
for these phenomena is that chlorophyll is present in a colloidal condition
in the cell. This point will be considered again later (see p. 85).

The Common Nettle (Urtica) is the plant which has been used for
material for the extraction of chlorophyll on a large scale, and it also
forms very useful material for extraction on a small scale. The pigment
has been found to be unaltered by drying, and, since dried leaves involve
far less bulk and dilution of solvents, material should be dried before
using. Some leaves (Elder and Conifers) are spoilt by drying. From
dried leaves pure solvents, such as petrol ether, benzene and acetone,
extract very little pigment for reasons which will be mentioned later,
but if the solvents contain a moderate amount of water, the pigment is
readily soluble. About 80°/o acetone is the best solvent. The nettle
leaves are removed from the stalks and laid on sheets of paper to dry.
When well air-dried they are finely powdered, and the powder further
dried at 30-40° C. in an incubator. The leaf-powder can be kept for a
considerable time in a well-stoppered bottle.

Expt. 18. Extraction of pigment. Two grains of leaf- powder are sucked to a filter-
paper on a small porcelain funnel and 2-3 c.c. of 85 % acetone are added. This is
allowed to soak into the powder for a few minutes. The fluid is then sucked through
with the pump. The operation is repeated until 20 c.c. of the solvent have been
added, when the powder is sucked dry. A deep blue-green solution with a red
fluorescence is obtained which contains all the four pigments from the leaf. The
acetone extract thus obtained is then poured into double the quantity of petrol ether
contained in a separating funnel. An equal quantity of distilled water is added, this

iv] CARBON ASSIMILATION 29

being poured gently down the side of the funnel in order to avoid the formation of
emulsions. In the course of a few minutes, the ether layer separates out and now
contains the pigments. The lower layer, which is slightly green, is run off. The
addition of distilled water and subsequent removal of the lower layer is repeated
about four times, in order completely to remove the acetone from the ether solution.
If the ether solution should have become at all emulsified, it can be cleared by
shaking with anhydrous sodium sulphate and filtering.

The whole process should be repeated with another 2 gms. of leaf-powder and the
pigment transferred to ether, since a solution in this solvent is required for later
experiments.

Expt. 19. Demonstration of the presence of chlorophylls a and b. Of the petrol
ether solution from the last experiment, 10 c.c. are shaken with 10 c.c. of 92 % methyl
alcohol. Two layers are formed of which the petrol ether layer contains chlorophyll
a, and the methyl alcohol layer chlorophyll 6. The solution of chlorophyll a is blue-
green, while that of chlorophyll b is a purer green, but the colour difference between
them is diminished owing to the presence of the yellow pigments, of which carotin
is in the petrol ether, and xanthophyll in the methyl alcohol.

As will be explained later, the green pigments of chlorophyll can be
saponified by alkalis and are then insoluble in ethereal solution. This
method can be adopted to separate the green from the yellow pigments,
xanthophyll and carotin.

Expt. 20. Separation of green and yellow pigments. Shake 5 c.c. of an ether
solution of the pigments (Expt. 18) with 2 c.c. of 30 % caustic potash in methyl
alcohol (obtained by dissolving 30 gms. of potassium hydroxide in 100 c.c. of methyl
alcohol !). After the green colour has reappeared, slowly add 10 c.c. of water and
then add a little more ether. On shaking the test-tube, two layers are produced, of
which the lower watery-alkaline one contains the saponified green pigments, while
the carotin and xanthophyll are contained in the upper ethereal layer.

Expt. 21. Separation of the two yelloiv pigments. The ether layer obtained in
the last experiment is washed with water in a separating funnel, and evaporated
down to 1 c.c. It is then diluted with 10 c.c. of petrol ether and next mixed with
10 c.c. 90% methyl alcohol. The methyl alcoholic layer is removed and the petrol
ether layer is again treated with methyl alcohol arid the methyl alcohol again
removed. This process is repeated until the methyl alcohol is no longer coloured.
The methyl alcohol contains the xanthophyll, the petrol ether the carotin.

Further accounts of the yellow pigments are given on p. 39.

The best known reactions of chlorophyll are those which take place
with acids and alkalies respectively.

Chlorophyll is a neutral substance, and, on treatment with alkalies,
it forms salts of acids, the latter being known as chlorophyllins. These
salts are soluble in water forming green solutions which are not however

1 The methyl alcohol must be very pure, otherwise the alcoholic potash solution will
become brown and discoloured.

30 CARBON ASSIMILATION [CH.

fluorescent. Chlorophyll a may be represented as the methyl phytyl
ester of an acid chlorophyllin (phytol is a primary alcohol, see p. 38):

/COOCHj ,COOH

C:>2H30ON4Mg/ C32H;wON4Mg<'

NCOOC.20H39 XCOOH

Chlorophyll a Chlorophyllin

On treatment in the cold with alkali, the ester is saponified, and the
alkali salt of chlorophyllin is formed. During saponification, there is a
change of colour in the pigment, the so-called brown phase, followed by
a return to green.

ExpL 22. Saponification of a mixture of the green pigments. Pour a little of the
ether solution obtained in Expt. 18 into a test-tube, and in a pipette take a little
30 % solution of potash in methyl alcohol. Place the lower end of the pipette at the
bottom of the test-tube and allow the potash to run in below the chlorophyll solu-
tion. At the interface between the solutions there appears immediately a brown -
coloured layer which diffuses on shaking. In about ten minutes it changes back
through an olive-green colour to pure green.

The chlorophyll has been saponified to the potassium salt of the acid chlorophyl-
lin. This salt is insoluble in ether, so if water is added to bring about a separation
of the two layers, the green colour is no longer present in the ethereal layer.

The change of colour on saponification is different for the two
chlorophylls, the brown phase produced in the above mixture of chloro-
phylls being due to a yellow phase produced by chlorophyll a, and a
brown-red phase produced by chlorophyll b. To demonstrate this the
phase test (Expt. 22) may also be carried out separately on the two
chlorophylls.

Expt. 23. Saponification of chlorophylls a and b separately. The methyl alcohol
.solution obtained in Expt. 19 is transferred to ether as in Expt. 18. Both the latter
and the petrol ether solution of chlorophyll a are saponified as in the previous
experiment.

As already demonstrated the potassium salts of the chlorophyllins
which are produced by saponification of the mixture of green pigments
in the cold are not fluorescent. By saponification of chlorophyll with
hot alkali, isochlorophyllins are formed (see Expt. 24 below) which are
fluorescent.

On heating chlorophyllins with concentrated alcoholic alkalies, a series
of decomposition products, phyllins (also acids) are obtained by removal
of carboxyl groups. The final phyllin has only one carboxyl group. When
this is removed, a substance, aetiophyllin, C31H34N4Mg, is obtained which
contains no oxygen (see Scheme 1, p. 34).

Another difference between the results of treating chlorophyll with
hot and cold alkali is that in the former process the yellow pigments are

iv] CARBON ASSIMILATION 31

<k-stroyed. If then water is added after saponification with hot alkali,
and the solution is shaken up with ether, the ether will remain colourless.

When chlorophyll is treated with acids, a different reaction takes
place. The chlorophyll changes in colour to olive-green and loses most
of its fluorescence. The magnesium of the molecule is removed, being
replaced by hydrogen, and the resulting product is termed phaeophytin
(see Scheme 1, p. 34).

From phaeophytin a series of decomposition products have been
obtained, which fall into two groups, the phytochlorins and the phyto-
rhodins. The phytochlorins are olive-green in colour, and are derived
from chlorophyll a: the phytorhodins are red, and are derived from
chlorophyll b. The phaeophytins from the two chlorophylls are indis-
tinguishable until the above decomposition products are obtained.
(The original discovery of two kinds of chlorophyll was brought about
bv the differentiation of these decomposition products.)

A number of phytochlorins and phytorhodins have been identified
and are designated by letters a, 6, etc. By employing a uniform method
of treatment, however, two of these products, phytochlorin e and phyto-
rhodin gy can be secured.

The phytochlorins and the phytorhodins are of course magnesium-free
compounds and can be obtained by the action of acid on the chlorophyllins
and isochlorophyllins. Phytochlorin e and phytorhodin g, in particular
are obtained by the action of acid on isochlorophyllins, i.e. they are'
magnesium-free isochlorophyllins. They are formed by the addition of
acid to the products of saponification with hot alkali.

The separation of the various phytochlorins and phytorhodins can be
brought about by means of their different distribution between ether
and hydrochloric acid : each compound can be extracted from ether
according to the concentration of the acid used.

Expt. 24. The formation of phytochlorin and phytorhodin. 5 c.c. of an ether
solution containing both chlorophylls a and b are evaporated to dryness in a test-
tube in a water-bath, and the residue treated with 3 c.c. of boiling 30 % potash
solution in methyl alec hoi, and boiled gently for half a minute. A liquid with a red
fluorescence is produced which consists of a solution of the potassium salts of
isochlorophyllins. The solution is diluted with double its volume of water, and
concentrated hydrochloric acid is added until the solution is just acid. The liquid is
then shaken with ether in a separating funnel : the dissociation products produced
by the previous treatment pass into the ether solution which thus acquires an
olive -brown colour.

The ether solution is shaken twice, each time with 10 c.c. of 4% hydrochloric
acid (sp. gr. 1'02 i.e. 12'9 c.c. strong acid: 87'1 c.c. water), and the green-blue acid
layer is separated and neutralized with ammonia and shaken with more ether, which

32 CARBON ASSIMILATION [CH.

then contains in solution phytochlorin e, the derivative df chlorophyll a. The phyto-
chlorin e gives to the ether an olive-green colour.

The ether layer remaining in the funnel, after the separation of the green-blue acid
layer, is now extracted with 10 c.c. of 12 °/0 hydrochloric acid>(sp. gr. 1*06 i.e. 38'1 c.c.
strong acid: 61 '9 c.c. water). The green acid solution so obtained is diluted with
water and shaken with ether which then becomes coloured red and contains phyto-
rhodin g, the derivative of chlorophyll b.

If the phyllins are acted upon by mineral acids, they lose their
magnesium in the same way as the chlorophyllins, and the series of
substances obtained in this way are termed porphyrins. Thus aetiophyllin
will give aetioporphyrin CsiH^N^j (see Scheme 1, p. 34).

The derivatives of chlorophyll which are free from magnesium, such
as phaeophytin, phytochlorin, phytorhodin, the various porphyrins, etc.
combine readily with the acetates of some metals such as copper, zinc
and iron, and they form intensely coloured, stable compounds. The change
of colour is so noticeable that the smallest traces of certain metals can
be detected in this way. Hence it is very difficult to prepare the
magnesium-free chlorophyll unless the reagents are perfectly pure and
all contact with certain metals is avoided.

Expt. 25. Substitution of copper for magnesium in chlorophyll. 2 c.c. of an ether
solution of chlorophyll are shaken with a little 20% hydrochloric acid (sp. gr. 1*10
i.e. 62'4 c.c. strong acid: 37'6 c.c. water), and then washed with water in a separating
funnel. In this way is produced in ether solution the magnesium -free chlorophyll
derivative, phaeophytin. The solution is evaporated down on a water-bath, and the
residue dissolved in 5 c.c. of alcohol. The solution is olive-green in colour. This is
heated and a grain of copper acetate or zinc acetate is added. The colour changes
back to a brilliant green, but without fluorescence (if all the chlorophyll has been
converted into phaeophytin).

From the results of these recent investigations, it is now possible to
write formulae for the two chlorophylls as follows :

chlorophyll a (C32H3oO N4Mg)
chlorophyll b (C32H28O2N4Mg) (COOCH3)

from which it will be seen that the phytol component amounts to one-
third of the weight of the chlorophyll.

From the analyses of chlorophylls from different plants, it was found
that the phytol content varied, and plants which yielded little phytol
most readily produced "crystalline chlorophyll," a form of the pigment
which has been known for some considerable time to previous workers.
The Cow Parsnip (Heradeum Sphondylium), Hedge Woundwort (Stachys
sylvatica) and Hemp-nettle (Galeopsis Tetrahit) are plants which readily
give crystalline chlorophyll. In this connexion it has been suggested

iv] CARBON ASSIMILATION 33

that the chlorophyll in plants is accompanied by an enzyme, chlorophyl-
lase, which, in alcoholic media, brings about alcoholysis of the chlorophyll,
and replaces the phytyl by the ethyl radicle. The products, formerly
known as crystalline chlorophyll, are now termed cnlorophyllides :

(CS2H3()ON4Mg)(COOCH3)(COOC20H39) + C2H5OH

= C2oH39OH + (C3,H30ON4Mg)(COOCH3)(COOC2H5).

Phytol Ethyl chlorophyllide

Similar chlorophy Hides are produced by other alcohols. In aqueous
solutions chlorophyllase brings about hydrolysis and the free acid
chlorophyllide is formed (see Scheme 2, p. 34):

(C32H3oON4Mg) (COOCH3) (COOC^) + H2O

= C2oH39OH +(C32H3oON4Mg) (COOCH3) (COOH).

Chlorophyllide

Chlorophyllase is a very stable enzyme ; it is not even destroyed by
boiling in alcohol for a short time, but if leaves are boiled in water, the
enzyme is destroyed.

Expt. 26. Microscopic examination of ethyl chlorophyllide. Prepare sections of
fresh Heracleum leaves and mount them in a drop of 90 % alcohol. Leave the slide
under a bell-jar containing a dish of alcohol. The section slowly dries in the course
of half a day or a day. It is then examined under the microscope when there svill be
observed the characteristic triangular and hexagonal crystals of ethyl chlorophyllide
(crystalline chlorophyll).

Expt. 27. Production of methyl chlorophyllide in the leaf. Sections may be used
as in the preceding experiment, or a piece of a leaf may be employed. In the latter
case a test-tube with 4 c.c. of 75 °/0 methyl alcohol is taken and 1 gm. of fresh leaf
is added to it. The leaf first becomes a darker green and then during the course of
a few hours becomes yellowish. On holding the leaf to the light there can be
observed with the naked eye a number of black points. If sections of the leaf be cut
and examined under the microscope, these spots appear as aggregates composed of
rhombohedral crystals, occurring in certain cells.

Expt. 28. Extraction of ethyl chlorophyllide. Two grams of dry Heracleum leaf-
powder are left for a day in a test-tube containing 6 c.c. of 90 % alcohol. The extract
is then filtered through a small porcelain funnel and the powder on the filter washed
with a little acetone. The filtrate is mixed with an equal quantity of ether, and then
with some water. The ether solution is transferred to a separating funnel and
thoroughly washed with water, and then concentrated on a water-bath to £ or 1 c.c.,
and 3 c.c. of petrol ether are added. On standing, the ethyl chlorophyllide is pre-
cipitated in the form of crystalline aggregates. It is freed from yellow pigments by
shaking with a little ether, and can be further purified by redissolving in ether and
precipitating again with petrol ether.

Expt. 29. The action of chlorophyllase. Fresh leaves of a species rich in chloro-
phyllase, e.g. Heracleum or Galeopsis, are finely divided and put in a 70 % acetone
solution, 3 c.c. of solution being used for every gram of leaf-powder. The chlorophyll,

o. 3

34

CARBON ASSIMILATION

[CH.

by means of the chlorophyllase, is hydrolyzed into phytol and the acid chlorophyllide.
This can be demonstrated after about a quarter of an hour if the solution is diluted
with water, transferred to ether and shaken with O05 °/0 sodium hydroxide. The
sodium hydroxide takes up more colouring matter the further the enzyme action
has progressed.

Expt. 30. The destruction oj chlorophyllase. If fresh leaves of a species rich in
chlorophyllase are first steeped in boiling water for a few minutes before they are
placed in the acetone solution, unaltered chlorophyll is extracted which does not
react with dilute alkali.

With acids — *-
(C32H30ON4Mg) (COOCH3) (COOC,0H39) - > (C32H32ON4) (COOCH,

chlorophyll a phaeophytin a

(C32H30ON4Mg) (COOH) (COOH) - *• (C32H32ON4) (COOH) (COOH)

chlorophyllin a phytochlorin e

and isochlorophyllin a and phytochlorins / and g

intermediate phyllins

intermediate porphyrins

aetiophyllin

Scheme 1.

aetioporphyrin

chlorophyll a
-joO) (COOCH3

with

methyl chlorophyllide a ^

(MgN4C32H300) (COOCH3) (COOCH3) £

chlorophyllide a
(MgN4C32H300) (COOCH3)(COOH)

dilute acid

with
dilute acid

with

dilute acid
Scheme 2.

phaeophytin a
(N4C32H320) (COOCH3) (COOCaoHs,)

t>>
tg'o

il

methyl phaeophorbide a
(N4C3.,H320) (COOCH3) (COOCH3)

phaeophorbide a
(N4C3.,H32O) (COOCH3) (COOH)

By treatment with acids, magnesium is removed from the chlorophyl-
lides, with the production of the corresponding phaeophorbides. Thus
methyl chlorophyllide a gives methyl phaeophorbide a, etc. (see
Scheme 2; above).

It has been previously mentioned that water-free solvents, such as
acetone, ether and benzene, in which pure extracted chlorophyll is

iv] CARBON ASSIMILATION 35

soluble, will not extract the pigment from thoroughly dried leaves, but
if a little water is added, it readily goes into solution. From fresh leaves
also these solvents can extract the pigments.

As an explanation of the above phenomena, it has been suggested
that chlorophyll in the chloroplastid is in the colloidal state, and that,
when water is added to the dried leaf, a solution of mineral salts in the
leaf is formed which alters the colloidal condition of the chlorophyll and
makes it soluble. This view is supported by the fact that if a colloidal
solution of chlorophyll in water, made from the pure extracted pigment,
is shaken with ether, the ether remains colourless. If, however, a little
salt solution is added and the mixture shaken, the ethereal layer
becomes green. In preparing the colloidal solution the solvent, acetone,
is replaced by the medium, water, in which chlorophyll is insoluble.

The condition of chlorophyll* is altered by plunging the leaves into
boiling water. The pigment is then much more readily soluble in ether,
etc., even when the leaves are subsequently dried. It is supposed that
the chlorophyll has diffused out from the plastids, and is in true solution
in accompanying waxy substances which have become liquid owing to
change of temperature.

Expt. 31. Preparation of a colloidal solution of chlorophyll. Take 10 c.c. of an
acetone extract of chlorophyll (Expt. 18) and pour this acetone solution into a large
volume of distilled water (100 c.c.), the liquid being continually stirred. This opera-
tion can be most conveniently done by taking the acetone solution in a pipette and
allowing it to run out of the pipette while the latter is used as a stirring rod in the
water. Note the change in colour to a purer green, and the disappearance of
fluorescence.

Expt. 32. To demonstrate the difference between a true and a colloidal solution of
chlorophyll. Evaporate 10 c.c. of an acetone extract (Expt. 18) to complete dryuess
and test its solubility in ether, petrol ether and benzene. It is soluble in all three
solvents. Now add these solvents to some of the colloidal solution prepared in the
last experiment, and note that the chlorophyll does not dissolve in any of these
solvents. If, however, some salt solution, e.g. a little magnesium sulphate, be added,
the chlorophyll is precipitated from its colloidal state and is now soluble in ether
and other solvents.

Expt. 33. To show that chlorophyll in the plant is probably in the colloidal
condition. Some nettle powder is carefully dried, e.g. by keeping it at 30^40° C. in an
oven, and then further drying in a vacuum desiccator over sulphuric acid. Small
quantities of this dry powder are put in test-tubes, and different pure water-free
substances such as acetone, ether, benzene and absolute alcohol are added. Note
that these solvents are not coloured by the chlorophyll. It can be demonstrated that
the extracted pigment is easily soluble in any of these substances. Repeat the experi-
ment with nettle powder moistened with a few drops of water, and note that the
solvents are immediately coloured.

3—2

36 CARBON ASSIMILATION [CH.

Expt. 34. Pure solvents are able to extract chlorophyll from fresh leaves. Crush
10 gms. of fresh leaves of nettle, horse-chestnut or elder in a mortar with some clean
sand, and put the crushed material on a filter-paper in a porcelain funnel. Add
20 c.c. of pure acetone and suck it through by means of a water-pump. Repeat this
several times. The pure solvent is here able to extract the pigment.

Expt. 35. Treatment of fresh leaves with boiling water changes the condition of
the chlorophyll. Dry a quantity of leaves which have been put in boiling water and
examine their solubility as in Expt. 33. Note that the chlorophyll in this powder is
soluble in pure solvents.

There is finally another change which chlorophyll can undergo,
namely that of allomerization, which takes place in alcoholic solution.
The characteristic of allomerized chlorophyll is that it does not give the
brown phase when treated with alkali (see Expt. 22). Allomerization is
accelerated in alkaline solution but inhibited by small quantities of acid.

Expt. 36. To demonstrate that allomerized chlorophyll does not give the brown
phase test. Dissolvte a little crude chlorophyll, obtained by evaporating an ether
solution, in absolute alcohol. To a sample of this add a little alkali, and perform the
phase test, from time to time, till at last the brown phase no longer appears.

CONNEXION OF CHLOROPHYLL WITH FORMALDEHYDE.

In addition to the above, another chemical property of chlorophyll
of great interest, is that connected with the production of formaldehyde.
Those investigators, who have sought to confirm the formaldehyde
hypothesis of carbon assimilation, have based their evidence on tests for
formaldehyde both in the plant and in chlorophyll-containing systems
outside the plant. By exposing films, or solutions, of chlorophyll to light
in presence of carbon dioxide, they have detected formaldehyde as a
result (Usher and Priestley, 5).

The most recent investigations (Jorgensen and Kidd, 2) have shown
that the experimental evidence is at present inadequate to support the
hypothesis, since formaldehyde arises from chlorophyll itself in the
absence of carbon dioxide.

In this later work (Jorgensen and Kidd, 2) on the behaviour of ex-
tracted chlorophyll in light, use has been made of a colloidal solution (see
p. 35) of pure chlorophyll (chlorophylls a and 6) for experimental work.
The solution for this purpose must be prepared from pure chlorophyll,
which has been tested and shown to be free from yellow pigments, since
the latter absorb oxygen and may confuse the issue of the experiment.
The pure chlorophyll is prepared by extracting dried nettle leaves with
80-85 °/0 acetone in the usual way and transferring to petrol ether (p. 28).

iv] CARBON ASSIMILATION 37

The petrol ether extract is then washed with 80 °/0 acetone to remove
colourless impurities, and with 80 °/o methyl alcohol to remove xantho-
phyll. Finally all traces of acetone and methyl alcohol are removed by
washing with water. This renders the chlorophyll insoluble in petrol
ether, since it is only soluble in this solvent if traces of other solvents
are present. Hence the pigment is precipitated out as a fine suspension,
leaving the carotin in solution. The chlorophyll is filtered off through
powdered talc, taken up in ether, reprecipitated by petrol ether and
finally obtained as a blue-black micro-crystalline substance. The col-
loidal solution or sol is made by dissolving 04 gm. of pure chlorophyll in
3 c.c. of absolute alcohol and pouring into 300 c.c. of distilled water.

The advantage of using such a solution is that the experimental
conditions, in all probability, approach more nearly to the conditions in
the plant, and reactions with other substances take place more readily
than when the chlorophyll is used as a film. The use of pure, instead of
crude, chlorophyll is also important as by this means it is possible to
determine the changes taking place in chlorophyll itself without complica-
tions arising from the accompanying impurities. The discordant results
of various workers on this subject are doubtless due to the employment
of crude chlorophyll. Ethyl alcohol is the best solvent for preparing
the sol since it does not produce formaldehyde when exposed to light
under ordinary circumstances in glass vessels. Methyl alcohol and
acetone should be avoided as they themselves either contain or give rise
to formaldehyde.

The chlorophyll sol is electro-negative. It is stabilized by weak
alkalies, but precipitated by weak acids.

Working with such a colloidal solution the results may be summarized
as follows.

When a chlorophyll sol is exposed to light in an atmosphere of
nitrogen in a sealed tube, no apparent change takes place in the chloro-
phyll, and no formaldehyde is produced.

When exposed in an atmosphere of carbon dioxide in a sealed tube,
the chlorophyll rapidly turns yellow- or brown-green. In the case of
sols of high concentration, the colour-change is preceded by precipitation
of the pigment. The same change takes place in the dark, only more
slowly. No formaldehyde is produced, and no absorption of carbon
dioxide could be detected. The yellow product has been shown to be the
magnesium-free derivative, phaeophytin, which is produced from the
pigment by the action of acids. The changes observed are explained by
the fact that the carbon dioxide, acting as a weak acid, first precipitates

.38 CAKBON ASSIMILATION [CH.

the sol, if concentrated, and then acts, like other weak acids, on the
chlorophyll, producing phaeophytin. If the solution is kept neutral by
addition of sodium bicarbonate, there is no colour change. The identity
of phaeophytin was shown by the spectrum and by the restoration of
colour on adding a trace of copper acetate.

When exposed to light, and the atmosphere in the sealed tube is
replaced by oxygen or air, the chlorophyll turns yellow- or brown-green
as before and then bleaches. The change of colour from green to yellow
or brown is again due to the formation of phaeophytin, this being brought
about by the presence of an acid substance, which is produced during
bleaching, and increases throughout the process. Formaldehyde can be
detected in a very slight amount during bleaching, but is formed in
much greater quantity after bleaching is complete.

It is suggested that the formaldehyde is produced by the oxidation
and breaking down of the phytol component of the chlorophyll :
CH3— CH— CH— CH— CH— CH— CH— CH— C = C— CHUOH

till ! I ! I

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

There is no reason for ascribing to any of the above reactions any
part in carbon assimilation. There is at present no hypothesis, supported
by satisfactory evidence, as to the process of carbon assimilation.

Expt. 37. Detection of formaldehyde as a product of oxidation of chlorophyll.
Extract 2 gms. of dried nettle leaf powder with 20 c.c. of 80 °/0 acetone and transfer
it to petrol ether as in Expt. 18. Then shake the petrol ether extract four or five
times with an equal volume of 80 % acetone to remove colourless impurities. Next
the petrol ether extract is similarly shaken up with 80% methyl alcohol which
removes the xanthophyll. This should be repeated until the methyl alcohol is
colourless. The petrol ether is finally washed repeatedly with water to remove traces
of acetone and methyl alcohol. The chlorophyll is in time precipitated as a fine
suspension, being insoluble in pure petrol ether. This suspension is filtered through
either kieselguhr or powdered talc on a small porcelain filter. The chlorophyll is
extracted from the powder on the filter with as small a quantity as possible of
absolute alcohol. This alcoholic solution is then poured, with constant stirring, into
100 c.c. of distilled water by which means a colloidal solution of chlorophyll is
obtained.

The test to be employed for formaldehyde is as follows (Schryver, 4). To 10 c.c. of
the liquid to be tested add 2 c.c. of a 1 % solution (freshly made) of phenylhydrazine
hydrochloride, 1 c.c. of a 5 % solution (freshly madej of potassium ferricyauide and
5 c.c. of concentrated hydrochloric acid. If formaldehyde is present a pink to
magenta colour is developed, either deep or pale, according to the quantity of form-
aldehyde. The reaction is due to the formation of a condensation product of
formaldehyde and phenylhydraziue, and this compound, on oxidation, yields a weak
base forming a coloured salt with concentrated hydrochloric acid. The salt is
readily dissociated again on dilution of the solution.

iv] CARBON ASSIMILATION 39

Two modifications (Schryver, 4) can be adopted in applying this test. First, in
testing for formaldehyde in pigmented solutions, the following course can be pursued.
The reaction mixture, after addition of phenylhydrazine, ferricyanide and hydro-
chloric acid, is diluted with water, and ether is added in a separating funnel. The
hydrochloride of the chromatogenic base is dissociated and the base is taken up by
the ether. The aqueous solution is run off, and on addition of strong hydrochloric
acid to the ether, the base passes into the acid as a coloured hydrochloride again.
By using a small quantity of acid, the sensitiveness of the test is increased, since the
colour is now distributed through a small quantity of liquid only.

The second modification consists in warming the solution to be tested for
a short time with the phenylhydrazine hydrochloride before adding the other
reagents. In this way, formaldehyde can also be detected if it should be in a poly-
merized form.

As a control, 10 c.c. of the colloidal solution of chlorophyll should be tested,
using both the above modifications. The remainder of the solution should be
exposed to sunlight (or the light from either an arc or mercury vapour lamp) in a
loosely corked vessel, until it is completely bleached. The bleached solution, on
testing, will be found to give a positive test for formaldehyde.

THE YELLOW PLASTID PIGMENTS.

These have already been mentioned in connexion with the leaf
pigments (pp. 28 and 29). In addition, however, they have a further
significance in that they constitute the pigments, located in plastids,
of most yellow and orange flowers and fruits. Sometimes also they occur
in other organs, i.e. root of Carrot (carotin).

Carotin, C^H^, is an unsaturated hydrocarbon. It crystallizes in
lustrous rhombohedra which are orange-red by transmitted and blue by
reflected light. It is readily soluble in chloroform, benzene and carbon
bisulphide, but with difficulty in petrol ether and ether.

One of its most characteristic properties is that it readily undergoes
oxidation in air, and becomes bleached. With concentrated sulphuric
acid it gives a deep blue colour.

Xanthophyll, C^H^O^ also forms yellow crystals with a blue
lustre. It is soluble in chloroform and ether, but insoluble in petrol
ether. It is more soluble than carotin in methyl alcohol. It gives
a blue colour with sulphuric acid, and also oxidizes in air with bleaching.

The separation of the two pigments (see Expt. 21) is based on the
fact that in a mixture of petrol ether and methyl alcohol containing a
little water, the carotin passes entirely into the petrol ether, whereas the
greater part of the xanthophyll remains in the methyl alcohol layer.

40 CARBON ASSIMILATION [CH. iv

REFERENCES
BOOKS

1. Willstatter, R., und Stoll, A. Untersuchungen iiber Chlorophyll.
Methoden und Ergebnisse. Berlin, 1913.

PAPERS

2. Jbrgensen, I., and Kidd, P. Some Photochemical Experiments with Pure
Chlorophyll and their Bearing on Theories of Carbon Assimilation. Proc. R. Soc.,
1917, B Vol. 89, pp. 342-361.

3. Jorgensen, I., and Stiles, W. Carbon Assimilation. A Review of Recent
Work on the Pigments of the Green Leaf and the Processes connected with them.
New Phytologist, Reprint, No. 10. London, 1917.

4. Schryver, S. B. The Photochemical Formation of Formaldehyde in Green
Plants. Proc. R. Soc., 1910, B Vol. 82, pp. 226-232.

5. Usher, F. L., and Priestley, J. H. A Study of the Mechanism of Carbon
Assimilation in Green Plants. I. Proc. R. Soc., 1906, B Vol. 77, pp. 369-376.
II. Ibid. 1906, B Vol. 78, pp. 318-327. III. Ibid. 1912, B Vol. 84, pp. 101-112.

CHAPTER V

CARBOHYDRATES AXD THEIR HYDROLYZING ENZYMES

THE carbohydrates which occur in plants may be classified as
follows :

f Pentoses, C5H10O5 — Arabinose, xylose.
Monosaccharides ...... JHexoses, C.HMO«— Glucose, galactose,

mannose, laevulose.

Disaccharides ......... {Sucrose, maltose,

T i isaccharides ......... {Raffinose and others.

f Pentosans, (C5H8O4)n — Araban, xylan.
Starches, (C6H10O5)n — Starch, dextrin, inulin.
Polysaccharides ...... ^ Mannans, galactans, gums, mucilages,

pectic substances.
Celluloses, (C6H10O5)n.

The carbohydrates are widely distributed in plants and form most
important parts of their structure. Those most commonly found are :
cellulose, starch, pentosans, dextrin, glucose, sucrose, laevulose, and
maltose. Other sugars, especially trisaccharides, are known in addition
to those mentioned above, but they are somewhat restricted and specific
in their distribution.

As in the case of the proteins, so with the carbohydrates, the molecules
of the more simple and soluble crystalline compounds, such as the
monosaccharides, are synthesized into more complex molecules which
exist, either in the colloidal (dextrin), or insoluble state (starch, cellulose).
The last-mentioned build up parts of the solid structure of the plant.
The resolution of the solid complex substances into simple ones is
known in many instances to be brought about in the plant by enzymes,
and it is highly probable that the synthesis of the complex from the
simple is also controlled by these enzymes.

The most commonly occurring sugars in plants are glucose, laevulose,
sucrose and maltose : sucrose is hydro! yzed by the enzyme, invertase,
into one molecule of glucose and one molecule of laevulose : maltose by
the enzyme, maltase, into two molecules of glucose. Both invertase and
maltase are widely distributed. The connexion between various sugars
and photosynthesis, and their inter-relationships with each other in the
leaves, are reserved for another section.

42 CARBOHYDRATES AND THEIR [CH.

Of the polysaccharides, cellulose is universally distributed in higher
plants and constitutes the greater part of the cell-walls. The pentosans,
galactans and mannans also, but to a lesser degree, are components of
their structure. Starch, in addition, is very widely distributed : it is
converted by the enzyme, diastase, into dextrin and maltose, and possibly
the same enzyme also controls its synthesis. In some plants no starch
is formed, and its place in metabolism is taken by inulin or cane-sugar.

The various carbohydrates will first be dealt with in detail, and later
their inter-relatioriships will be considered.

MONOSACCHARIDES.

These are termed tetroses, pentoses or hexoses according to the
number of carbon atoms in the molecule. They contain primary
(— CH2OH) or secondary (= CHOH) alcohol groups, and either an
aldehyde (— CHO) group, as in glucose, or a ketone (= C = O) group, as
in laevulose. They are, as a class, white crystalline substances, soluble
in water and aqueous alcohol, but insoluble in ether, acetone and many
other organic solvents. They are capable of certain characteristic
chemical reactions which form a basis for their detection and estimation.
One of the most important is that connected with the aldehyde and
ketone groups, owing to which they act as reducing agents, being them-
selves oxidized. The reducing action usually employed is that which
takes place with copper salts in hot alkaline solution, whereby cuprous
oxide is formed. Hence they are termed "reducing" sugars. Another
important reaction is the formation of crystalline osazones (only in the
case of sugars with aldehyde or ketone groups), which, by virtue of
their melting points and characteristic crystalline forms, constitute, in
several cases, valuable tests for the presence of sugars.

PENTOSES.

These sugars contain five carbon atoms, and have the general formula
C5H10O5. They are said to be present in the free state to some extent
in leaves (Davis and Sawyer, 10). In plants they occur chiefly, however,
as condensation products formed with elimination of water. These
products are termed the pentosans, and are widely distributed ; on
hydrolysis they yield pentoses again. The various gums found in plants
consist largely of pentosans, and the pectins also contain pentose groups;
both consequently yield pentoses on hydrolysis (see pp. 61 and 64).

HYDROLYZING ENZYMES

If we examine the structural formula of a pentose, as for example,

arabinose :

H— C = 0

HO— C*— H
H— C*— OH
H— C*— OH

!

H— C— H
OH

we see that each of the three carbon atoms marked * is united to four
different atoms or groups of atoms. Each of these carbon atoms is there-
fore asymmetric, and, with regard to it, there are two possible isomers
(see p. 9, Cole, 3, for stereoisomerism). It will be found on examination
that there are eight possible isomers of the formulae given above :

CHO

HO— C— H
H— C— OH

CHO

CHO

CHO

1
HO— C— H

1
H-C— OH

1
H— C— OH

HO— C— H

H— C— OH

1
HO— C— H

HO— C— H

I

CH2OH
Z-Ribose

CHO

I
H— C— OH

HO— C— H
HO— C— H

CH2OH

/-Arabinose

H— C— OH

CH2OH
c?-Ribose

CHO

HO— C— H
H— C— OH
H— C— OH

CH2OH

d- Arabinose

H— C— OH

|

CH2OH
/-Xylose

CHO

H— C— OH

I
H— C— OH

I
HO— C— H

CH2OH

£-Lyxose
unknown

CH2OH
d- Xylose

CHO

HO— C— H
HO— C— H

—OH

CH2OH
d-Lyxose

Of these only seven have been isolated. The two pentoses which
occur in plants are /-arabinose and /-xylose. These, however, are known
almost solely as condensation products, pentosans, in gums, woody tissue,
etc. The pentoses form osazones (see p. 49 for reactions and composition).

Arabinose. This sugar occurs as the pentosan, araban, in various
gums, such as Cherry Gum, Gum Arabic, etc. (see p. 45).

Some of the properties and reactions of the pentoses are demonstrated
in the following experiments.

44 CARBOHYDRATES AND THEIR [CH.

Expt. 38. Tests for arabinose. For reactions a-e use a 1 % solution of arabinose :
for reaction f a 0*2 °/0 solution.

If pure arabinose is not available, a solution for tests a, b and c can be prepared
from gum arabic. Boil 5 gms. of the gum in 100 c.c. of water with 10 c.c. of con-
centrated hydrochloric acid for 5 minutes and then neutralize to litmus with alkali.
Such a solution is only suitable for the specific tests for arabinose, since it also
contains galactose (see p. 61). For tests a, b and c small pieces of solid gum arabic
may even be used.

(a) Heat a few c.c. of the sugar solution in a test-tube with about half its volume
of concentrated hydrochloric acid. In the mouth of the test-tube place a piece of
filter-paper soaked with aniline acetate (made by mixing equal quantities of aniline,
water and glacial acetic acid). A pink colour will be produced in the paper. This is
due to the fact that furfural is formed by the action of the acid on the pentose, and
the furfural then gives a red colour with aniline acetate solution :

;OH Hj

I I CH = CH

CH— CH-iOHi \

| .OH ;— 3H20 ^>0

CH-C< CH = C

i I Nc=o \r 0

JOH "Hi | . ° = °

H H

Arabinose Furfural

This reaction, however, is also given by the hexoses but to a much less
extent.

(b} Warm a few c.c. of the sugar solution with an equal volume of concentrated
hydrochloric acid in a test-tube, and add a small quantity of phloroglucin. A bright
red coloration is produced.

(c) To a few c.c. of the sugar solution in a test-tube add an equal quantity of
concentrated hydrochloric acid, and then a little solid orcinol. Divide the solution
into two equal portions. Heat one portion. The solution will become red changing
to violet and finally blue, blue-green or green. To the other portion, after heating for
a time, add a few drops of 10% ferric chloride solution. A deep green colour is at
once produced. In both cases, on the addition of a little arnyl alcohol, the green
colour will be extracted by the alcohol.

(d) a-Naphthol reaction. Add to a little of the sugar solution a few drops of a
1 % solution of a-naphthol in alcohol. Mix the two solutions and then run in about
5 c.c. of concentrated sulphuric acid down the side of the test-tube. A violet colora-
tion is produced at the junction of the two liquids. The coloration is due to a
condensation product of a-naphthol with furfural, the latter being formed by the
action of the apid on the carbohydrate. This reaction is likewi.se given by laevulose
and cane-sugar (since it yields laevulose, see p. 52), and less strongly by glucose and
maltose ; also by some proteins which contain a carbohydrate group.

(e} Boil a little of the arabinose solution with a few drops of Fehling's solution.
Reduction will take place.

( /*) Make the osazone of arabinose following the instructions given for glucosazone
(see p. 49).

v] HYDROLYZING ENZYMES 45

A solution of arabinose which will give the pentose reactions can also
be obtained by hydrolysis of Cherry Gum. The gum oozes from the bark
of various species of Prunus, such as the Cherry (Primus Cerasus) and
the Bird Cherry (P. Padus).

Expt. 39. Preparation of arabinose solution from Cherry Gum. The gum is heated,
on a water-bath in a round-bottomed flask fitted with an air condenser1, with
dilute sulphuric acid (1 pt. by wt. of gum : 7 pts. by wt. of 4 °/0 sulphuric acid) for
about 5 hours. The solution is then neutralized with calcium carbonate and filtered.
Perform the tests a, 6 and c of Expt. 38 on the solution. A positive result is obtained
in each case. Since the solution contains other sugars as impurities, it cannot con-
clusively be used for tests c?, e and/. If a considerable quantity of gum is available,
crystallization of arabinose should be attempted by concentrating the aqueous sugar
solution, extracting this with 90 % alcohol and again concentrating in a desiccator
(see p. 53). If a very small quantity of gum only is available, the tests a, b and c
should be performed directly on a small piece of the gum in a test-tube.

A purer preparation of arabinose, which may be used for all the tests
of Expt. 38, can be obtained by the hydrolysis of araban (see Expt. 48).

Xylose. This sugar occurs very widely distributed in woody tissue as
the pentosan, xylan (see p. 53). A solution of xylose which will give the
pentose reactions can be obtained from the hydrolysis of straw.

Expt. 40. Preparation of xylose solution from straw. Take about 50 gms. of straw,
which has b*een cut up into small pieces, and put it into a round- bottomed flask
fitted with an air condenser. Add sufficient 5 °/0 sulphuric acid to cover the straw
and heat on a water-bath for 2-3 hrs. Filter off the solution, neutralize with
calcium carbonate and filter again. Make with the solution the tests a and c of
Expt. 38. The solution will also reduce Fehling's solution strongly, but this reduction
may be partly due to other sugars formed in the hydrolysis.

The presence of xylan giving the pentose reactions can also be demonstrated in
straw, bran or sawdust by merely heating small quantities of these substances in a
test-tube with the above reagents (see Expt. 49).

A purer solution of xylose can be obtained from the hydrolysis of
xylan (see Expt. 51).

\Vherj xylose is oxidized with bromine, it yields xylonic acid which
has a characteristic cadmium salt. The formation of this salt is used as a
method for identifying the sugar (see Expt. 51).

HEXOSES.

Glucose. This substance, which is also known as grape-sugar, is very
common and very widely distributed in plants. It occurs in the tissues
of leaves, stems, roots, flowers and fruits. It is produced as a result of

1 i.e. a wide piece of glass tubing about 3 ft. long passing through the cork.

46

CARBOHYDRATES AND THEIR

[CH.

the hydrolysis of cane-sugar and maltose, and, in all probability, is the
first sugar synthesized from carbon dioxide and water. Its synthesis and
its relationships to other sugars will be discussed later (see p. 69). It is
a white crystalline substance, readily soluble in water and aqueous
alcohol, but only slightly soluble in absolute alcohol.

If, as in the case of a pentose, we examine the structural formula for

a hexose, such as glucose :

H— C = O

H— C*— OH
HO— C*— H
H— C*— OH
H— C*—

OH

H— C— H
OH

we see that there are four carbon atoms marked * which are united
to four different groups of atoms. It will be found in this case that there
are sixteen possible isomers :

CHO

H— C— OH
H— C— OH

HO— C— H
HO— C— H

CH2OH
£-Mannose

CHO

HO— C— H
HO— C— H
H— C— OH
H— C—

OH

CH2OH

d-Mannose

CHO

HO— C— H

HO— C— H

HO-

CH.2OH

^-Glucose

H— C— OH

CH2OH

rf-Glucose

CHO

HO— C— H

H— C— OH
HO— C— H

H— C— OH

CH2OH
2-Idose

CHO
H— C— OH

HO— C— H

I
H— C— OH

HO— C— H
CHOH

CH2OH

Z-Gulose

I
CHO

I
HO— C— H

HO— C— H

H— C— OH

j
HO— C— H

CH2OH

rf-Gulose

HYDROLYZING ENZYMES

47

CHO

H— C— OH

HO— C— H

H— C— OH
HO— C— H

CH2OH

l-Galactose

HO — C — H
H— C— OH

CH2OH

c?-Galactose

CHO
H— C— OH

H— C— OH

I
H — C — OH

HO— C— H

CH2OH

Z-Talose

CHO

HO— C— H
HO— C— H
HO— C— H
H— C— OH

JH2OH
d-Talose

CHO

HO— C— H

I
HO— C— H

HO— C— H

I
HO— C— H

CH2OH

CHO

H— C— OH

H— C— OH

H— C— OH

H— C— OH

CH,OH

HO— C— H

HO — C— H
HO— C— H

;H,OH

These four unknown

CHO

HO— C— H
H— C— OH

-OH
-OH

CH2OH

Though many of the above sugars have been synthesized artificially,
only three are known to occur naturally, i.e d-glucose (dextrose or grape-
sugar), d- man nose and c£-galactose.

Since compounds containing asymmetric carbon atoms are optically
active, i.e. can rotate a plane of polarized light, it follows that the sugars
under discussion are optically active.

d-glucose is dextro-rotatory.

When either d- or Z-glucose is first dissolved in water, it is chemically
less active than would be expected of the aldehyde form depicted above.
This is explained by assuming that glucose, when first dissolved in water,
exists in the condition of a 7-lactone :

CH2OH

48

CARBOHYDRATES AND THEIR

[CH.

In the above state the carbon atom marked * is also asymmetric so
that two forms of glucose are possible, a- and /3-glucose :

HO—

HO--C— H

H— C

CH2OH

a-Glucose

In solution, both the above forms pass by tautomerism into the
aldehyde form.

In the plant there are, as will be described later (p. 142), many aromatic
and other compounds containing one or more hydroxyl groups. These
hydroxyl groups of the aromatic substances are frequently replaced by
a glucose (or other sugar) molecule, and such compounds are termed
glucosides, as, for instance, salicin, the glucoside of salicylic alcohol which
occurs in Willow bark (see p. 152) :

H— C

CH2OH

Salicin

These substances, moreover, may be classified either as a- or /3-
glucosides according to which of the above a or /3 forms of glucose
combine with the residual part of the compound. Various glucosides will
be dealt with in Chaps, vn and IX.

Expt. 41. Tests for glucose. Before dealing with the sugars actually isolated from
the plant, it is advisable that the following tests and reactions should be performed
with pure glucose using a 0'2 °/0 solution.

(«) Moore's test. Boil a little of the glucose solution with an equal volume of
caustic soda solution. A yellow colour is developed which is due to the formation of
a condensation product (caramel) of the sugar.

v] HYDROLYZING ENZYMES 49

(6) Trom^ier's test. Add a few drops of a 1 °/0 copper sulphate solution to 2-3 c.c.
of 5 % caustic soda solution. A blue precipitate of cupric hydroxide is formed. Add
now 2-3 c.c. of the glucose solution, and the precipitate will dissolve. On boiling,
the blue colour disappears, and a yellow or red precipitate of cuprous oxide is formed.
If only a little sugar is present the blue colour will disappear, but no oxide may
be formed.

(c) Fehling's test. Boil a few c.c. of freshly made Fehling's solution in a test-tube
and note that it is unaltered. Then add an equal quantity of the glucose solution
and boil again. A red precipitate of cuprous oxide is formed.

(cT) Osazone test. Take 10 c.c. of a 0'5 °/0 solution of glucose in a test-tube and
add as much solid phenylhydrazine hydrochloride as will lie on a sixpenny piece,
at least twice as much solid sodium acetate and also 1 c.c. of strong acetic acid.
Warm gently until the mixture is dissolved and filter into another test-tube. Then
place the tube in a beaker of boiling water for at least ^ hour, keeping the water
boiling all the time. Let the test-tube cool slowly, and a yellow crystalline deposit
of phenylglucosazone will separate out. Examine this under the microscope and it
will be found to consist of fine yellow needles variously aggregated into sheaves and
rosettes. Glucosazone melts at 204-205° C.

The osazone reaction takes place as follows :

CH2OH(CHOH)4CHO + H2N-NHC6H.5 = CH2OH (CHOH)4CH : N -NHC6H5 + H2O.

Glucose phenylhydrazone

The phenylhydrazone is very soluble, but if an excess of phenyl-
hydrazine is used, a second hydrazine complex is introduced and an
insoluble osazone is formed :

CH2OH (CHOH)3— C— CH : N • NHCGH5

II
N-NHC6H6

Glucose reacts in this way by virtue of its aldehyde group. Phenyl-
hydrazine hydrochloride does not give an osazone when boiled with
glucose unless excess of sodium acetate be added. This acts on the
hydrochloride to form phenylhydrazine acetate and sodium chloride.

Galactose. Galactose rarely, if ever, occurs free in plants, though it
is fairly widely distributed in the form of condensation products, the
galactans, in combination with other hexoses and with pentoses (see p. 60).
These galactans form constituents of various gums, mucilages, etc.
Agar-agar, which is a mucilage obtained from certain genera of the Red
Seaweeds (Rhodophyceae), yields a high percentage of galactose on
hydrolysis with acids. Galactose also occurs as a constituent of some
glucosides from which it may be derived on hydrolysis.

One of the most important reactions of galactose is the formation of
mucic acid on oxidation with nitric acid. Mucic acid is practically
insoluble in water and separates out as a crystalline precipitate on
pouring the products of oxidation into excess of water.

o. A.

50 CARBOHYDRATES AND THEIR [CH.

Expt. 42. Preparation of galactose from agar-agar. Weigh out 50 gms. of agar-
agar. Put it into a round- bottomed flask fitted with an air condenser (see p. 45).
Add 500 c.c. of 2 % sulphuric acid and heat on a water-bath for 4 hrs. Neutralize
the solution with calcium carbonate and filter. Concentrate on a water-bath to a
syrup. On standing, crystals of galactose will separate out. Then add a little 50-75 °/0
alcohol and warm gently on a water-bath. By this means much of the dark-coloured
product will go into solution and can be poured off leaving the crystalline residue.
Take up this residue in a little hot water, boil well with animal charcoal to decolorize
the solution and filter. . Concentrate again on a water-bath. On cooling, colourless
prisms of galactose will separate out.

Expt. 43. Oxidation of galactose to mucic acid. Heat the galactose obtained in
the last experiment with nitric acid (1 gm. galactose to 12 c.c. of nitric acid of sp. gr.
1*15, i.e. 5 pts. of concentrated acid and 12 pts. of water) on a water-bath, until the
liquid is reduced to one-third of its bulk. Then pour the product into excess of
distilled water. On standing (for a day or two), a white sandy microcrystalline preci-
pitate of mucic acid will separate out.

Mannose. Mannose has not been detected free in many plants, but
is widely distributed as condensation products, the mannans, in certain
mucilages and in the cell-walls of the endosperm of various seeds
(see p. 59). From the mannans the sugar can be obtained by hydrolysis.
On adding phenylhydrazine hydrochloride and sodium acetate to a
solution of mannose, the phenylhydrazone, which is nearly insoluble in
water, is formed almost immediately and hence constitutes a ready
method for the detection of the sugar.

Laevulose. This sugar, which is also termed fructose, is widely
distributed in plants, in the tissues of leaves, stems, fruits, etc. It is
formed, together with glucose, in the hydrolysis by acids of cane-sugar.
The original cane-sugar is dextro-rotatory, whereas laevulose is more
laevo-rotatory than glucose is dextro-rotatory ; hence the mixture from
the hydrolysis is laevo-rotatory and is known as invert sugar, the change
being termed inversion. The same hydrolysis is brought about by the
widely distributed enzyme, invertase. The polysaccharide, inulin, also
yields laevulose on acid hydrolysis. Laevulose is a white crystalline
substance, soluble in water and alcohol. Unlike glucose, it contains a
ketone instead of an aldehyde group :

CH2OH

C = 0

I
HO— C— H

H-C— OH
H— C— -OH

CH2OH
c?-Fructose

v] HYDROLYZING ENZYMES 51

Laevulose reduces Fehling's and other copper solutions. It yields
the same osazone as glucose with phenylhydrazine hydrochloride and
sodium acetate. It also forms an osazone with methylphenylhydrazine
(m.p. 158° C.), a reaction which constitutes a distinction from glucose
since the latter gives no osazone with this substance.

Expt. 44. Tests for laemtlose. The following tests should be performed with a
0*2 % solution of laevulose in the same way as for glucose (see p. 48).
(a) Moore's test. A positive result is obtained.
(6) Trommels test. A positive result is obtained.

(c) Fehling's test. Reduction takes place.

(d) Osazone test. Note that the crystals are identical with those formed from
glucose.

(e) a-Naphthol test (see p. 44). A strong reaction is given.

(/) Seliwanof's test. To 5 c.c. of SeliwanofFs solution (prepared by dissolving
0*05 gm. of resorcinol in 100 c.c. of 1 in 2 hydrochloric acid) add a few drops of
laevulose solution and boil. A red coloration and a red precipitate are formed. Add
a little alcohol and the precipitate forms a red solution.

DlSACCHARIDES.

These sugars are formed from the monosaccharides by condensation
with elimination of water. By boiling with dilute acids, or by the
action of certain enzymes, they are hydrolyzed into monosaccharides.
The two most important disaccharides found in plants are maltose and
cane-sugar.

Maltose. Maltose or malt-sugar, though it probably occurs in smaller
quantities than glucose and laevulose, is widely distributed in plant
tissues. It is formed in the hydrolysis of starch, and its relationships in
the plant to starch and to other sugars will be considered later. It is a
white crystalline substance soluble in water and alcohol. In constitution
it is a glucose-a-glucoside :

C6Hn05— O— C— H

HO— C— H

H— C— OH

CH2OH

Maltose

'it reduces Fehling's solution; but less readily than glucose. With
phenylhydrazine hydrochloride and sodium acetate it forms an osazone

4—2

52 CARBOHYDRATES AND THEIR [CH.

(m.p. 206° C.), which is more soluble than glucosazone and crystallizes
in broader flatter needles. Maltose is dextro-rotatory.

Expt. 45. Tests for maltose. The tests a, b, c and e should be performed with a
0*2 °/0 solution of maltose ; test d with a 2 % solution (see also glucose, p. 48).

(a) Moore's test. A positive reaction is given.

(b) Trommer's test. A positive reaction is given.

(c) Fehling's test. Reduction takes place, but less strongly than with glucose.

(d) Osazone test. Take 10 c.c. of the solution and treat as for glucosazone. The
crystals of maltosazone will be found to be much broader than those of glucosazone.

(e) Hydrolysis. Take 20 c.c. of the sugar solution and add 2 c.c. of concentrated
hydrochloric acid. Heat in a boiling water-bath for half an hour. Neutralize and test
for the osazone. Glucosazone will be formed.

Sucrose. Sucrose or cane-sugar is very widely 'distributed in plants,
in leaves, stems, roots, fruits, etc. It is a white substance which crystal-
lizes well, and is soluble in water and alcohol. As previously stated it is
hydrolyzed by dilute acids and by invertase into one molecule of glucose
and one molecule of laevulose. It is formed by the condensation of
glucose and laev^ose with the elimination of water. Its constitution is
in all probability as follows :

o

CH,OH • C • (CHOH)2 ' CH/

3

CH -(CHOHVCH • CHOH CH,OH

so that both the ketone and aldehyde groups are rendered inactive. It
does not reduce Fehling's solution and does not form an osazone. It is
dextro-rotatory.

Expt. 46. Tests for cane-sugar. The following tests should be made with a 1 °/n
solution of pure crystalline cane-sugar (see also glucose, p. 48).

(a) Moore's test. A negative result is obtained.

(6) Fehling's test. No reduction takes place.

(c) a-Naphthol test. A positive result is given since sucrose yields laevulose.

(d} Hydrolysis. To a few c.c. of the solution add a drop of strong sulphuric acid
and boil for two minutes. Then neutralize with caustic soda using litmus as
indicator. Boil again and add Fehling's solution drop by drop. A reduction takes
place owing to the inversion of the cane-sugar by sulphuric acid.

(e) Seliwanoffis test. A positive result is [obtained owing to the liberation of
laevulose.

v] HYDROLYZING ENZYMES 53

POLYSACCHARIDES.

These substances are formed by condensation, with elimination of
water, from more than three molecules of monosaccharides.

PENTOSANS.

It has already been mentioned that condensation products of the
pentoses, the pentosans, are widely distributed. The two most frequently
occurring pentosans are xylan and araban. No enzymes are known
which hydrolyze thejDentosans. It is characteristic of xylan and araban
that they form copper compounds in Fehling's solution in presence of
, excess of alkali.

Araban. This pentosan may be regarded as a condensation product
of arabinose as already indicated. It occurs in various gums (Gum
Arabic, Cherry Gum) frequently in combination with other substances.
On hydrolysis with acids, araban yields arabinose. (See also gums and
arabinose.)

Expt. 47. Preparation of araban from Gum Arabic. (Salkowski, 27.) Weigh out
20 gms. of gum arable and^dissolve in 500 c.c. of warm water in a large evaporating
dish. Then add 200 c.c. of Fehling's solution and excess of caustic soda solution.
f The araban will^e precipitated as a white gtrfnmy mass which will settle at the
bottom of the dish. "Filter off' through muslin. Take up the precipitate in dilute hydro-
chloric acid (1 pt. of acid : 1 pt. of water), and then add alcohol. The araban separates
out as a white precipitate. Wash away the copper chloride with alcohol.

Expt. 48. Hydrolysis of araban. The araban from the last experiment is put
into a round-bottomed flask with about 200 c.c. of 2 °/0 sulphuric acid and heated on
a water-bath for 2 hours, the flask being fitted with an air condenser (see p. 45).
Then neutralize the liquid with calcium carbonate, filter from calcium sulphate, and
concentrate on a water-bath. The sugar is extracted from the syrup with 90 % alcohol.
Arabinose crystallizes with difficulty but thev process may be facilitated by sowing
the concentrated alcoholic solution with a few crystals of arabinose. Some of the
solution of arabinose should be tested with all the tests given in Expt. 38.

Xylan. This pentosan occurs in lignified cell-walls, and is the chief
constituent of "wood gum." It is found in the wood of many trees (not
Coniferae), in bran, in wheat and oat straw, in maize cobs, in the shells
of coconuts and walnuts, in the testa of the cotton (Gossypium) and in
many other tissues : also in some gums. On hydrolysis, xylan yields
xylose; hence wood shavings, bran, straw, etc., will give the pentose reac-
tions on hydrolysis.

54 CARBOHYDRATES AND THEIR [CH.

Expt. 49. Detection of pentose from pentosans in bran, sawdust and straw. Take
a small quantity of bran and boil it up several times with 98 % alcohol, filtering off
the alcohol after each treatment. This should remove any sugars or glucosides
present. Allow the alcohol to evaporate off' from the bran, and then make the
following tests for pentoses (see Expt. 38) :

(a) Heat, for about one minute, a small quantity of the bran in a test-tube, with
sufficient concentrated hydrochloric acid to cover it. Care should be taken not to
char the material. Then add as much solid orcinol as will lie on the tip of a pen-
knife. Heat gently again for a few seconds. Then add one or two drops of strong
ferric chloride solution ; a green coloration will be produced. Add amyl alcohol and
the green colour will pass into the alcohol.

(6) Heat again another portion of the bran with the same quantity of concentrated
hydrochloric acid in a test-tube, but this time heat more strongly. After heating a
few minutes place a piece of filter-paper soaked in a solution of aniline acetate in the
mouth of the test-tube. A cherry-red coloration will denote the formation of
furfural.

The above method and tests with bran may be repeated in exactly the same way
using sawdust or straw.

Expt. 50. Preparation of xylan from sawdust. Extract one kilo of sawdust with
4 litres of 1-2 % ammonia solution for 24 hrs. Then filter off" the ammoniacal solution
through muslin and repeat the extraction. The xylan is insoluble in ammoniacal
solution, and in this way colouring matters are removed. Finally wash the sawdust
well with water and press dry from the liquid. Then add to the sawdust sufficient
5% caustic soda solution to make a thick mush (about 1000-1500 c.c.) and allow it
to stand for 24 hrs. in a warm place. The alkaline solution is then pressed out
through calico and filtered through filter-paper. To the clear filtrate add an equal
volume of 96 °/0 alcohol which will precipitate the xylan as a sodium compound.
Filter off this precipitate, wash with alcohol, and decompose with alcohol to which
a little strong hydrochloric acid has been added to remove the sodium. The free
xylan is again washed with alcohol, and can be dried by washing with absolute
alcohol and ether and finally in a desiccator. It is a dirty-white powder which is
almost insoluble in water. Make the tests for pentoses (see Expt. 38) on a little of
the solid xylan. The reaction will be given in each case.

Expt. 51. Hydrolysis of xylan. Put the xylan obtained in the last experiment
in a round-bottomed flask fitted with an air condenser (see p. 45). Add 100 c.c. of
4% sulphuric acid and heat on a water-bath for 4 hrs. Neutralize the solution with
calcium carbonate, filter from calcium sulphate and concentrate on a water-bath.
Test a portion for pentoses (see Expt. 38) and a positive reaction will be obtained.
To a small quantity add also a few drops of Fehling's solution and boil. Reduction
will take place.

To the remainder of the xylose solution add bromine (see p. 45) gradually until
there is excess. Then remove the excess of bromine by warming on a water-bath.
Neutralize the solution, which contains xylonic acid, with cadmium carbonate and
evaporate on a water- bath. Extract the residue with alcohol and filter. On concen-
trating the alcoholic extract, white prismatic needles of cadmium xylonate separate
out.

v] HYDROLYZING ENZYMES 55

It has been shown that pentosans, xylan and probably araban, occur
in leaves (Davis, Daish and Sawyer, 15). It is likely that the xylan is
widely distributed in all tissues since it forms a constituent of lignified
cell-walls.

Expt. 52. Detection of pentoses from pentosans in leaves. (Davis, Daish and
Sawyer, 15.) Take two large leaves of the Sunflower (Efelianthus annuus). Tear into
small pieces and drop into boiling 98 % alcohol in a flask. Boil well and filter off the
alcohol. Eepeat until all the green colour is removed. Then dry off the alcohol and
grind up the leaf residue. Perform the test for pentoses (Expt. 38 a and c) on the
dry leaf tissue. It should give the above tests showing the presence of pentosans.

Leaves of the Violet (Viola odorata] and Nasturtium (Tropaeolum majus] may
also be used.

Expt. 53. Method for determination of pentosans in tissues, bran and leaves, etc.
Weigh out 2 gins, of bran, put it into a round-bottomed flask, add 100 c.c. of 12 %
hydrochloric acid and fit the flask with a water condenser. Heat gently over wire
gauze and distil into a solution of phloroglucin in 12 % hydrochloric acid. A green
precipitate of furfural phloroglucide is formed which eventually becomes almost
black. For accurate estimations of pentosans this is filtered off" and weighed on a
Gooch crucible. The same method may be used with leaf residue as in Expt. 52.

STARCHES.

Starch. This is a very widely distributed substance in plants. It
occurs as solid grains throughout the tissues, in leaves, stems, roots,
fruits and seeds. It is absent, however, from a number of Monocotyle-
dons, e.g. Iris, Snowdrop (Galanthus), Hyacinihus, etc. It forms one of
the chief reserve materials of plants, that is, it is synthesized from sugar
when carbon assimilation and carbohydrate synthesis are in progress, and
is stored in the solid form in tissues as grains. In other circumstances of
the plant's existence, when material for metabolism is not available
from carbon assimilation, as for instance in germinating seeds or
growing bulbs or rhizomes, the starch is hydrolyzed into dextrin and
soluble sugar, which is translocated and used as a basis for meta-
bolism. During the night in leaves there is also a similar hydrolysis
of the starch wThich has been temporarily stored from the excess of
sugar synthesized during the day.

Starch has a very large molecule and thus a high molecular weight. It
is insoluble in cold water. When heated with a little water it gives starch
paste, but on boiling with water it gives an opalescent ''solution" which
really contains starch in the colloidal state as an emulsoid. In this con-
dition it does not diffuse through dialyzing membranes and does not
depress the freezing point of water. The " solution " cannot, strictly

56 CARBOHYDRATES AND THEIR [CH.

speaking, be filtered, but generally, when hot, it passes to some extent
through ordinary filter-paper. Starch is insoluble in alcohol and is pre-
cipitated by it.

The most characteristic reaction of starch is the blue colour it gives
with iodine solution. This blue colour disappears on heating, but re-
appears again on cooling. Starch is precipitated from "solution" by
half saturation with ammonium sulphate : it does not reduce Fehling's
solution.

By boiling with dilute acids, starch is first converted into " soluble
starch " which still gives a blue colour with iodine. On further boiling,
various dextrins (see dextrins) are obtained which give either purple,
red or no colour with iodine. The final product, after prolonged boiling
with acids, is glucose. Hydrolysis with diastase yields dextrin and
maltose (see diastase, p. 73).

Expt. 54. Preparation of starch from Wheat. Starch may be prepared from
a cereal by the following method.

Take 25 gms. of flour and make it up into a dough with a little water. Allow it
to stand for half an hour. Then tie a piece of muslin over the top of a beaker which
is filled with water. Place the dough on the top of the muslin and rub it gently with
a glass rod. The starch will be separated from the gluten, and will be washed
through the muslin and on standing will sink to the bottom of the beaker. Allow
this to stand till the starch has settled, then decant off the bulk of the liquid. Filter
off the starch, and wash well with water, then with alcohol and finally with ether.
Dry in the steam-oven.

For the detection of starch in green leaves, see Expt. 77.

Expt. 55. Tests for starch. Take a small quantity of the starch prepared in the
previous experiment and shake up with a little cold water in a test-tube. Filter, and
test the filtrate with a drop of iodine (in potassium iodide) solution. No blue colour
is obtained. Pour a drop of the iodine solution on the residue in the filter. It turns
deep blue.

Weigh out 2 gms. of the starch prepared in the last experiment, and mix it into
a thin cream with a little water. Boil rather more than 100 c.c. of water in an evapo-
rating dish, and then gradually add to it the starch paste, keeping the water boiling
all the time. An opalescent " solution " is obtained. With a few c.c. of the solution
in each case make the following tests:

(a) Add 1-2 drops of iodine solution. A blue colour is obtained. Heat the solu-
tion : the blue colour disappears, but reappears on cooling.

(6) Add an equal volume of alcohol : the starch is precipitated.

(c) Add an equal volume of saturated ammonium sulphate solution : the starch
is precipitated, i.e. by half saturation with this salt.

(d) Add basic lead acetate solution : the starch is precipitated.

Expt. 56. Hydrolysis of starch. To 50 c.c. of the starch solution prepared in the
last experiment add 1 c.c. of strong sulphuric acid. Boil for 10-20 minutes in a

v] HYDROLYZING ENZYMES 57

round -bottomed flask. Test a portion of the solution with iodine from time to time ;
a purple, red or brown colour is formed due to the dextrin produced in hydrolysis.
To the remainder of the solution after neutralization, using litmus as indicator, add
some Fehling's solution and boil. Reduction takes place owing to the glucose formed
in hydrolysis.

DEXTRINS.

These compounds occur in the plant as transitory substances, since
they are formed as intermediate products of the hydrolysis of starch by
diastase. They are also formed on heating starch or by boiling it with
mineral acids (see previous experiment). The hydrolysis of starch to
dextrins is fairly rapid, but the conversion of dextrins into maltose is
a much slower process.

Both starch and dextrins have the same empirical formula. Various
forms of the latter have been identified, such as amylodextrin which gives
a blue colour with iodine, erythrodextrin which gives a brownish-red
colour with iodine, and achroodextrin which gives no colour with iodine.
The dextrins are readily soluble in water; they are precipitated by
alcohol but not by basic lead acetate. On hydrolysis with acids, they
are converted into glucose.

Expt. 57. Preparation of dextrin by hydrolysis of starch, (a) By diastase from
leaves of the Pea (Pisum sativum). Weigh out lOgms. of commercial potato starch
and make it into a solution in 250 c.c. of boiling distilled water as in Expt. 55 and
cool. Then weigh out 10-15 gms. of fresh leaflets of the Pea (Pisum sativum) and
pound them well in a mortar. Add to the pounded mass 100 c.c. of distilled water
and a few drops of chloroform (see maltase, p. 75) and filter. The filtrate will contain
diastase (see also Expts. 78-80). Then add the diastase extract to the starch solu-
tion in a flask, plug with cotton- wool and put in an incubator for 48 hrs. If a little
of the liquid is withdrawn from time to time and tested with iodine, it will be found
that the blue colour due to starch gradually disappears and is replaced by the
brownish-red colour due to dextrin. After 48 hrs. there will be no trace of blue
colour ; then filter the liquid and concentrate the filtrate on a water-bath to a syrup.
Treat the residue with 96-98 °/0 alcohol and filter. A sticky mass of dextrin is left
which should be extracted with a little hot alcohol and then reserved for the next
experiment. To show the presence of maltose, the alcoholic extract is evaporated to
dryness on a water-bath, the residue taken up in a little water and the osazone test
made (see p. 49) with the solution. Crystals of maltosazone will separate out.

(b) By diastase from germinating Barley (Hordeum vulgare). Grind well 25 gms.
of barley grains in a coffee-mill. Put the flour into a flask and extract with 96-98%
alcohol by heating on a water-bath. This will largely free the grain from sugars.
Make a starch " solution" of the residue by boiling with 500 c.c. of water and filtering
through fine muslin.

58 CARBOHYDRATES AND THEIR [CH.

Weigh out another 25 gms. of barley grains and allow them to germinate by
soaking and spreading on damp blotting-paper for 5-7 days. Pound the grains well
in a mortar, add 100 c.c. of water, allow to stand for 2-3 hrs. and filter. Precipitate
the filtrate with alcohol and allow to stand for 24 hrs. Filter off the precipitate,
take up in water and add it to the barley starch "solution," together with a few
drops of chloroform. Proceed as with (a) only the time for hydrolysis may be much
shorter, i.e. 6-12 hrs.

Expt. 58. Tests for dextrin. Make a solution of the dextrin prepared in the last
experiment and note that it is very soluble in water. With the solution make the
following tests :

(a) Add a little iodine solution. A reddish-brown colour is produced. Heat the
solution and the colour will disappear. Cool again and the colour will reappear.

(b) Add an equal volume of strong alcohol. The dextrin is precipitated.

(c) Add an equal volume of saturated ammonium sulphate solution, i.e. half
saturation with ammonium sulphate. The dextrin is not precipitated.

(d) Add some basic lead acetate solution: the dextrin is not precipitated.

INULIN.

Inulin. This substance occurs as a soluble "reserve material" in the
cell-sap of the underground stems, roots and also leaves of a number of
plants, especially members of the Compositae, e.g. Dahlia (Dahlia
variabilis), Jerusalem Artichoke (Helianthus tuber osus), Chicory (Cicho-
rium Intybus) and the Dandelion (Taraxacum officinale). It is said to
occur also in the Campanulaceae, Lobeliaceae, Goodeniaceae, Violaceae
and many Monocotyledons (Hyacinthus, Iris, Muscari and Scilla).

Inulin is a condensation product of laevulose to which it bears much
the same relation as starch to glucose. It is a white substance, soluble
in water and insoluble in alcohol. It crystallizes out in the cells, in
which it occurs, in characteristic sphaero-crystals on addition of alcohol
to the tissues. It is hydrolyzed by mineral acids to laevulose : also by
the enzyme inulase which occurs in the plant.

Expt. 59. Extraction of inulin. Cut off the tubers from two Dahlia (Dahlia
variabilis} plants, wash well, and put them through a mincing machine. Carefully
collect the liquid and the crushed tuber, and boil well with sufficient water to cover
the crushed material. Add also some precipitated calcium carbonate to neutralize
any free acids present. Then filter through fine muslin, and to the filtrate, which
should again be made quite hot, add lead acetate solution until a precipitate
(of mucilaginous substances, etc.) ceases to be formed. Care should be taken to
avoid the addition of a large excess of lead acetate. Filter off the lead precipitate,
and saturate the filtrate with sulphuretted hydrogen till all excess lead is removed.
Filter off" the lead sulphide, neutralize the filtrate to phenolphthalein with ammonia,
and evaporate to half bulk or less on a water-bath, when the inulin will probably

v] HYDROLYZING ENZYMES 59

begin to deposit, Then pour into an equal volume of alcohol, and allow to stand for
one or two days. The crude precipitate of inulin is filtered off, dissolved in a small
amount of water, and reprecipitated with alcohol. It can be washed with alcohol
and ether and dried over sulphuric acid.

The Artichoke (Helianthus tuberosus) may also be used, about 12 tubers being
necessary.

Expt. 60. Tests for inulin. Make a solution of some of the inulin prepared in
Expt. 59 in hot water. It will readily dissolve giving a clear solution. With the
solution make the following tests :

(a) Make a very dilute solution of iodine and add to it a drop or two of iuulin
solution : the brown colour will be unaffected.

(6) Boil some inulin solution with a little Fehling : no reduction takes place.

If the inulin solution which is being used should reduce Fehling it indicates that
sugar is present as impurity. If this is the case, then a little of the solid inulin
should be washed free from sugar by means of alcohol before proceeding with the
following tests.

(c) To a little inulin solution add some 1 % alcoholic solution of a-naphthol and
a few drops of concentrated sulphuric acid and warm. A deep violet colour is
produced. This is due to the formation of furfural from the laevulose produced in
hydrolysis (see laevulose, p. 51).

(d) To a little inulin solution add about an equal quantity of strong hydrochloric
acid and a few crystals of resorcin. A red coloration is formed. This reaction
(Seliwanoff 's test) is also due to the presence of laevulose (see laevulose, p. 51).

Expt. 61. Hydrolysis of inulin. Some inulin is dissolved in very dilute hydrochloric
acid (about 0'5 %) and heated on a water-bath for half an hour in a round-bottomed
flask provided with an air condenser (see p. 45). The solution is then neutralized
with sodium carbonate and concentrated on a water-bath. With the concentrated
solution make the following tests :

(«) Boil with a little Fehling : the solution is rapidly reduced.

(6) Make the osazone test (see p. 49). Glucosazone crystals will be found to be
formed on microscopic examination. (Laevulose forms the same osazone as glucose.)

(c) Make the tests (c) and (d] of the last experiment. A positive result will be
given in each case.

MANNANS.

The mannans which have already been mentioned (see p. 50) are
condensation products of the hexose, mannose. They occur most fre-
quently, either mixed, or in combination, with the condensation products
of other hexoses and pentoses (glucose, galactose, fructose and arabinose)
as galacto mannans, glucomannans, fructomannans, mannocelluloses, etc.
Such mixtures or compounds of which mannans form a constituent are
widely distributed in the seeds of many plants, i.e. Palms (including the
Date-palm), Asparagus (Ruscus), Clover (Trifoliuin), Coffee Bean (Coffea
arabica), Onion (Allium Cepa) and of members of the Legurninosae,

60 CARBOHYDRATES AND THEIR [CH.

Rubiaceae, Coniferae and Umbelliferae. In seeds the mannans may
constitute, together with cellulose, the thickened cell-walls of the endo-
sperm and are included in the term " reserve- or hemi-cellulose " though
they are not strictly celluloses. " Vegetable ivory," which is the endo-
sperm of the Palm, Phytelephas macrocarpa, contains considerable
quantities of a mannan and is used as a source of mannose. Mannans,
in addition, form constituents of certain mucilages, as for instance those
in Lily bulbs (Lilium candidum, L. bulbiferum, L. Martagon and others)
(Parkin, 23) and tubers of various genera of the Orchidaceae : they are
also found in the roots of the Dandelion (Taraxacum), Helianthus and
Chicory, Asparagus and Clover, and in the wood and leaves of various
trees.

Many of the mannans, unlike true celluloses, are readily hydrolyzed
by dilute hydrochloric and sulphuric acids. The mannan in the Coffee
Bean, however, is hydrolyzed with difficulty.

GALACTANS.

These substances bear the same relationship to the hexose, galactose,
as the mannans to mannose, that is, they are condensation products of
galactose (see p. 49). Similarly they frequently occur, together with
the condensation products of other sugars, as galactoaraban, galactoxylan,
galactomannan, etc. As such they form constituents of many gums and
mucilages and of the cell-walls of the reserve tissue of seeds, i.e. the
Coffee Bean (Goffea arabicd), the Bean (Faba), the Lupin (Lupinus), the
Paeony (Paeonia), the Kidney Bean (Phaseolus), the Date (Phoenix),
the Pea (Pisum), the Nasturtium (Tropaeolum) and many others (Schulze,
Steiger and Maxwell, 29).

GUMS.

These substances occur widely distributed among plants, especially
trees. Some gums are wholly soluble in water giving sticky colloidal
solutions : others are only partially soluble. They are all insoluble in
alcohol. In the solid state they are translucent and amorphous.

Chemically the gums are varied in nature ; they may in general be
regarded as consisting of complex acids in combination1 with condensa-
tion products of various sugars, such as araban, xylan, galactan, etc.
On hydrolysis they give mixtures of the corresponding sugars, arabinose,
xylose, galactose, etc., in varying proportions, though in some cases one
sugar preponderates.

v] HYDROLYZING ENZYMES 61

Some of the best-known gums are the following :

Gum Arabic (arabin). This substance is obtained from an Acacia
(Acacia Senegal), a native of the Soudan. The gum exudes from the
branches. Other species of Acacia yield inferior gums. Gum arabic is
a mixture of the calcium, magnesium and potassium salts of arabic acid,
a weak acid of which the constitution is unknown, in combination with
araban and galactan.

Gum Tragacanth. This is a product from several Tragacanth shrubs
which are species of Astragalus (Leguminosae), chiefly A. gummifer. It
is obtained by wounding the stem and allowing the gum to exude and
harden. On hydrolysis it gives a mixture of complex acids and various
sugars such as arabinose, galactose and xylose.

Cherry Gum (cerasin) occurs in the wood of the stems and branches
of the Cherry (Prunus Cerasus), the Bird Cherry (P. Padus), the Plum
(P. domestica), the Almond (P. Amygdalus) and other trees of the
Rosaceae. It exudes from fissures of the bark. On hydrolysis it yields
almost entirely arabinose.

Expt. 62. Reactions of Gum Arabic. Put a little gum arabic into an evaporating
dish and add a little water. Heat gently and stir. The gum will slowly dissolve,
giving a thick sticky solution which does not solidify or gel on cooling. Make the
following tests, using a little of the gum solution in a test-tube each time.

(a) Add a little alcohol. The gum is precipitated.

(6) Add a little Fehling's solution and boil. No reduction takes place.

The three following experiments show the presence of pentosan complexes in the
gum (see also Expt. 38, p. 44):

(c) Add a little phloroglucin to the gum and then strong hydrochloric acid. No
colour is produced. Now heat, and a cherry-red colour appears.

(cT) Heat the gum solution with a little concentrated hydrochloric acid and then
add a trace of orcinol. Warm again and then add one or two drops of strong ferric
chloride solution. A green coloration will be produced.

(e) Heat the gum solution strongly with hydrochloric acid, and, after heating for
a few minutes, place a piece of niter-paper soaked in a solution of aniline acetate in
the mouth of the test-tube. A cherry-red coloration indicative of furfural will be
formed.

Expt. 63. Hydrolysis of Gum Arabic. Weigh out 10 gms. of gum arabic. Put it
into a round-bottomed flask and add 100 c.c. of water and 4 c.c. of strong sulphuric
acid. Warm gently until the gum goes into solution. Then fit the flask with an
air condenser (see p. 45) and heat on a water-bath for about 4 hrs. Cool the
solution, and neutralize with barium carbonate. Filter off" the barium sulphate and
concentrate the solution on a water-bath. Boil a drop or two of the syrup with
Fehling's solution and show that reduction takes place. (The original gum either
does not reduce Fehling at all, or, if so, only slightly.) Then add a little nitric acid

62 CARBOHYDRATES AND THEIR [CH.

(sp. gr. 1'15, see Expt. 43) to the syrup and heat on a water-bath almost to dryness.
Pour the residue into about 100 c.c. of water and allow to stand. A microcrystalline
precipitate of mucic acid is formed showing the presence of galactose (see p. 50) as
a product of hydrolysis.

MUCILAGES.

The characteristic of these substances is that they swell up in water
and produce colloidal solutions which are slimy.

Mucilages are widely distributed and may occur in any organ of the
plant. Sometimes they are confined to certain cells, mucilage sacs or
canals. They are distinguished from the pectic substances by the fact
that they do not gelatinize. Some of the best known examples of
mucilage-containing tissues are those in the root and flower of the
Hollyhock (Althaea rosed) : in succulent plants (Aloe, Euphorbia), in
bulbs (Scilla, Allium) and tubers (Orchis Morio): in seeds of Flax or
Linseed (Linum) and in fruits of Mistletoe (Viscum album}.

The mucilages vary in composition. They appear to be largely, if
not wholly, condensation products of various sugars (galactose, mannose,
glucose, xylose, arabinose), similar constituents to those of many gums
and hemicelluloses. On hydrolysis various mixtures of sugars are pro-
duced. Of the mucilages, that from linseed has been thoroughly
investigated. It has been found on hydrolysis to give sugars only, e.g.
arabinose, xylose, glucose and galactose. In this respect mucilages differ
from gums, since the latter have always some other accompanying sub-
stance in addition to sugars.

Expt. 64. Preparation and properties of mucilage from Linseed (Linum) (Neville,
21). Take about 60 gms. of linseed and let it soak for 24 hrs. in 300 c.c. of water.
Then separate the slime from the seeds by squeezing through muslin, and add to the
liquid about twice its volume of 96-98 % alcohol. The mucilage is precipitated as
a thick slimy precipitate. Filter off the precipitate and wash with alcohol. By
washing with absolute alcohol and ether and finally drying in a desiccator, the
mucilage may be obtained as a powder.

Add water to some of the mucilage. It swells up and finally gives an opalescent
solution. Make with it the following tests :

(a) Add iodine. No colour is given.

(b) Add a little Fehling's solution and boil. No reduction takes place.

Expt. 65. Hydrolysis of Linseed mucilage. Put the remainder of the mucilage in
a round-bottomed flask and add 50 c.c. of 4 % sulphuric acid. Fit the flask with
an air condenser (see p. 45) and heat for at least four hours on a water-bath.
Cool and neutralize with barium carbonate. Filter off the barium sulphate, and

v] HYDROLYZING ENZYMES 63

concentrate the filtrate on a water- bath. With the concentrated solution make the
following tests :

(a) Add a few drops to a little boiling Fehling solution. Reduction immediately
takes place.

(6) Make the phloroglucin, orcinol and furfural tests for pentoses, using a small
quantity only of the hydrolysis mixture for the tests. A positive result will be given
in each case. The pentoses, arabinose and xylose, are responsible for these reactions.

(c) Add to some of the solution phenylhydrazine hydrochloride, sodium acetate
and a little acetic acid, and leave in boiling water for half an hour for the osazone test
[see Expt. 41 (d)\ A mixture of osazones will separate out, among which glucosazone
can be identified.

(d) Concentrate the remainder of the solution and then add some nitric acid of
sp. gr. T15 (see Expt. 43). Evaporate down on a water-bath to one-third of the bulk
of the liquid and then pour into about 100 c.c. of water. A white microcrystalline
precipitate of mucic acid will separate out, either at once or in the course of a day or
two. This demonstrates the presence of galactose.

PEC TIC SUBSTANCES.

These substances are considered at this point since they are said to
constitute, in more or less intimate connexion with cellulose, the middle
lamella of cell-walls in many tissues. The pectic substances are fre-
quently found in the juices of succulent fruits in which the tissues have
disintegrated, such as red currants and gooseberries. They have been
isolated chiefly from fleshy roots, stems or fruits, as, for instance, from
turnips, beetroot, rhubarb stems, apples, cherries and strawberries.

Recent work (Schryver and Haynes, 28) points to the fact that in
turnips, strawberries, rhubarb stems and apples, there is the same pectic
material, and it is possible that all such substances may be identical.
The compound isolated in the above case is of an acidic nature and has
been termed pectinogen. When pectinogen is treated with dilute
solutions of caustic alkali at ordinary temperatures, it is rapidly changed
into a second substance termed pectin, which is readily converted into
a gel under certain conditions.

In the case of juicy fruits, such as currants and gooseberries, the
pectinogen can be precipitated as a gelatinous precipitate by adding
alcohol to the expressed juice. In the case of fleshy fruits, stems and
roots, the procedure is as follows. The tissues are thoroughly dis-
integrated in a mincing machine and pressed free from all juice in a
powerful press. The residue is then dried, finely ground, washed with
water and finally extracted with dilute ammonium oxalate solution in
which pectinogen is soluble. The extract is concentrated and the

64 CARBOHYDRATES AND THEIR [CH.

pectinogen precipitated by alcohol. It may be purified by reprecipita-
tion.

Pectinogen is precipitated from aqueous solution by alcohol as a very
bulky gelatinous mass, but when dried it forms an almost colourless granu-
lar powder. Put into water it absorbs large quantities of liquid and dis-
solves slowly, giving an opalescent solution with a distinctly acid reaction.

As mentioned above pectinogen in alkaline solution is rapidly con-
verted into pectin. A solution of pectinogen is not precipitated either
by acid or dilute solutions of calcium salts but, after treatment with
alkali and conversion into pectin, both the aforesaid reagents produce
gelatinous precipitates. A similar precipitate is also formed when lime
water is added in excess to a solution of pectinogen and it is allowed to
stand. There is little doubt that the pectinogen is converted by the
alkali into pectin. Pectin is also an acid substance and it is insoluble
in water, giving an insoluble salt with calcium. After treatment of
pectinogen with alkali the pectin can, as already stated, be precipitated
by adding acid.

Analyses of pectin from various sources have led to the suggestion
of C^H^Oje as its formula. There is also evidence that it contains one
pentose group. This can be detected and estimated by the furfural
phloroglucide method (see Expt. 53).

Expt. 66. Extraction and reactions of pectinogen. Take about half a pound of red
currants and squeeze out the juice through fine muslin into a large beaker. Then add
to the juice about 2-3 times its bulk of 96-98% alcohol. A bulky gelatinous precipi-
tate of pectinogen will separate out. Allow the precipitate to stand for a time in the
alcohol, and then filter off. Wash with alcohol and finally press free from liquid.
Dissolve the precipitate in as little water as will enable it to go into solution. To two
small portions of the solution add respectively (a) a few drops of strong hydrochloric
acid, (6) an excess of calcium chloride solution. Note that no precipitate is formed in
either case.

Expt. 67. Conversion of pectinogen into pectin, and reactions of pectin. Take about
one-third of the pectinogen solution prepared in Expt. 66, make it alkaline with
caustic soda, and let it stand for about 10-15 minutes. Then divide the solution
into two parts and add respectively (a) sufficient hydrochloric acid to acidify,
(6) excess of calcium chloride solution. In the first case a gel of pectin is formed :
in the second case a gelatinous precipitate of the calcium salt of pectin.

To a further quantity of the pectinogen add excess of lime water and let it stand.
The gelatinous calcium precipitate will separate out in a short time.

Expt. 68. Detection of the pentose group in pectinogen. Filter off the pectin gel
obtained in the last experiment and allow it to dry. Then test for the pentose group
by the orcinol, phloroglucinol and furfural tests (see Expt. 38). All results will be
found to be positive.

v] HYDROLYZING ENZYMES 65

The extraction of pectinogen, etc. in the above experiments can equally well be
carried out with other material, e.g. ripe gooseberries, raspberries and strawberries,
using exactly the same methods.

Expt. 69. Preparation of pectinogen from Turnips. Take two full-sized turnips
and mince them finely in a mincing machine. Then wrap the mass in a piece of
strong unbleached calico and press out the juice as completely as possible in a press.
The juice contains little pectinogen and can be thrown away. The pressed mass is
then thrown into 0'5 °/0 ammonium oxalate solution heated to 80-90° C. on a water-
bath and stirred to make a paste. The liquid is again rapidly pressed out in the press.
To the viscid extract an equal volume of 96 % alcohol is added, and the pectinogen
separates out as a voluminous gelatinous precipitate. This is filtered off and, when
pressed free from alcohol and dried, can be used for tests as in the previous experi-
ments.

The gelatinization of pectinogen can also be brought about by certain
enzymes termed pectases which are found in the juices of various plants,
i.e. root of Carrot (Daucus Carota) and leaves of Lucerne (Medicago
sativa), Lilac (Syringa vulgaris) and Clover (Trifolium pratense).

Expt. 70. Action of pectase on pectinogen. Make an extract of either Lucerne or
Clover leaves by pounding them in a mortar with a little water, and then filter. Add
the filtrate to some of the pectinogen solution prepared in Expt. 66 or 69. On
standing a gelatinous precipitate will be produced. Should the reaction be slow, it
may be accelerated by placing the mixture in an incubator.

CELLULOSES.

Celluloses are very important polysaccharides. They form constituents
of the structural part of all the higher plants. The cell-wall of the
young cell consists entirely of cellulose, but in older cells the walls may
be lignified, cuticularized, etc., i.e. the cellulose may be accompanied by
other substances such as lignin, cutin, mucilage, etc. In the light of
these facts the term cellulose is made to include :

1. Normal celluloses.

2. Compound celluloses.

(a) Ligno-celluloses.
(6) Pecto-celluloses.
(c) Adipo- or cuto-celluloses.

3. Pseudo- or Reserve celluloses.

True or normal cellulose. Of this substance, as we have said,
many cell- walls are composed. The most familiar form of cellulose is
cotton, which consists of hairs, each being a very long empty cell, from
the testa or coat of the seed of the Cotton plant (Gossypium herbaceum).

o. 5

66 CARBOHYDRATES AND THEIR [CH.

Crude cotton (i.e. the hair cell-walls) is not quite pure cellulose, but
contains a small amount of impurity from which it is freed by treatment
first with alkali and subsequently with bromine or chlorine. All kinds
of cotton material, cotton-wool, and the better forms of paper (including
filter-paper) may be regarded as almost pure cellulose.

Pure cellulose is a white, somewhat hygroscopic, substance. It is
insoluble in water and all the usual solvents for organic substances. It
is, however, soluble in a solution of zinc chloride in hydrochloric acid in
the cold, and in a solution of zinc chloride alone on warming. It is also
soluble in ammoniacal cupric oxide (Schweizer's reagent).

In addition cellulose is soluble in concentrated sulphuric acid, which
on standing, converts it first into a hydrate and then finally into
glucose. If, however, water is added to the sulphuric acid solution as
soon as it is made, the gelatinous hydrate of cellulose is precipitated.
This substance is termed " amyloid " since it gives a blue colour with
iodine. Concentrated nitric acid converts cellulose into nitrates, of
which one is the substance, gun-cotton. In 10 °/0 alkalis cotton fibres
thicken and become more cylindrical. This procedure has been em-
ployed by Mercer to give a silky gloss to cotton, and the resultant
product is called mercerized cotton.

Expt. 71. The colour tests and solubilities of cellulose.

(a) Dip a little cotton-wool into absolution of iodine in potassium iodide. Then
put the stained wool into an evaporating dish and add a drop or two of concentrated
sulphuric acid. A blue coloration is given. This is due to the formation of the
hydrate " amyloid " mentioned above.

(6) Dip some cotton-wool into a calcium chloride iodine solution. (To 10 c.c. of
a saturated solution of calcium chloride add 0'5 gm. of potassium iodide and O'l gm.
of iodine. Warm gently and filter through glass-wool.) A rose-red coloration is
produced which eventually turns violet.

(c) Heat a strong solution of zinc chloride (6 pts. of zinc chloride to 10 pts. of
water) in an evaporating dish and add 1 part of cotton-wool. The cellulose will in
time become gelatinized, and if a little water is added from time to time, a solution
will eventually be obtained on continuous heating.

(d) Make a solution of zinc chloride in twice its weight of concentrated hydro-
chloric acid and add some cotton- wool. The wool will rapidly go into solutiQii in the
cold.

(e) Add some cotton-wool to an ammoniacal copper oxide solution and note that
it dissolves. (To a strong solution of copper sulphate add some ammonium chloride
and then excess of caustic soda. Filter off the blue precipitate of cupric hydroxide,
wash well, dry thoroughly, and dissolve in strong ammonia.) Add strong hydrochloric
acid and the cellulose is precipitated out again. Then add water and wash the
precipitate until it is colourless. Test the roughly dried precipitate with a little
iodine and strong sulphuric acid. A blue coloration is given.

v] HYDROLYZING ENZYMES 67

All the above tests may be repeated with threads from white cotton material,
with filter-paper and good white writing paper.

Try tests (a) and (6) with newspaper, and note that they are not so distinct as
with writing paper owing to the presence of ligno-cellulose (see Expt. 73).

Expt. 72. Hydrolysis of cellulose by acid. Dissolve as much filter-paper as possible
in 5 c.c. of concentrated sulphuric acid and when all is in solution pour into 100 c.c.
of distilled water. Boil the solution in a round-bottomed flask fitted with an air
condenser (see p. 45) and use a sand-bath for heating. After boiling for an hour,
cool and neutralize the solution with solid calcium carbonate. Add a little water if
necessary and filter. Test the filtrate with the following tests :

(a) Make the osazone [see Expt. 41 (c?)]. Note that crystals of glucosazone are
formed.

(6) Add a little Fehling's solution and boil. . Note that reduction takes place.

Instead of using filter-paper, the above experiment may also be carried out with
cotton-wool or threads from white cotton material.

Ligno-cellulose. As the cells in plants grow older the walls usually
become lignined, that is part of the cellulose becomes converted into
ligno-cellulose. The extreme amount of change is found in wood. The
least amount in such fibres as those from the stem of the Flax (Linum
mitatissimum) which, when freed from such impurities, consist of cellu-
lose only and constitute linen. Other fibres, containing more ligno-
cellulose, are those of the stem of the Hemp plant (Cannalris sativa)
and the Jute plant (Corchorus) from which string, rope, canvas, sacking
and certain carpets are made. The percentages of pure cellulose in
these various lignified tissues are as follows :

Cotton fibre 88'3%

Flax and Hemp fibre ... 72-73 %

Jute 54%

Beech and Oak wood ... 35-38 %

The ligno-celluloses are generally regarded as consisting of cellulose
and two other constituents, of which one contains an aromatic nucleus
and the other is of the nature of a pentosan (see xylan, p. 53). Both
are sometimes classed together and termed lignin or lignon. The lignin
reactions (see below) depend on the presence of an aromatic complex.
It has been suggested that coniferin, vanillin and allied compounds
which are present in wood are probably the substances responsible for
the reaction (Czapek, 6).

Although the best paper is made from cellulose, cheaper forms of
paper are manufactured from ligno-cellulose, and, as a result, they give
reactions for lignin and are also turned yellow by exposure to light.

Expt. 73. Reactions of lignin.

One of the most striking reactions of lignin (due as it is supposed to a furfural

5—2

68 CARBOHYDRATES AND THEIR [CH.

grouping) is the magenta-red coloration given by phloroglucin in the presence of
concentrated hydrochloric acid.

Soak the tissue to be experimented upon with an alcoholic solution of phloro-
glucin and then add a drop or two of strong hydrochloric acid. The magenta-red
colour will be produced.

As material, practically any lignified tissue may be used. Shavings from twigs
of any tree or shrub, e.g. pith and wood from the Elder (Sambucus nigra), will be
found useful : also shavings from a match ; straw, bran, coarse string, cheap white
paper, such as newspaper or white and pale-coloured papers used for wrappings.

Make the phloroglucin test on good white writing paper. It should not give the
reaction since it is made from cellulose.

Other phenols and their derivatives will also give colour reactions with lignin in
the presence of hydrochloric acid, but the colorations in most cases are not so much
developed as with phloroglucin (Czapek, 6). For this reason (though it is also possible
to use any of the lignified tissues suggested above) good results are obtained by using
strips of any cheap newspaper, since the reagents seem to penetrate this material
quickly.

Soak strips of newspaper (or other material) in alcoholic solution of the following
substances, or such of them as are available, and then add a few drops of concen-
trated hydrochloric acid. It is useful to put the material on a white glazed tile or

plate :

Reaction

Phenol blue-green coloration

Resorcinol ... violet ,,

Orcinol ... ... red- violet „

Catechol ... greenish-blue „

Pyrogallol ... blue-green „

Guaiacol ... yellow-green „

Cresol greenish „

a-Naphthol ... greenish „

Thymol ... green „

Indol ... ... cherry-red „

Skatol cherry-red „

It should be noted that strong hydrochloric acid alone will sometimes
give a red colour with woody tissues: this is due to the presence of
phloroglucin in the wood itself (see phloroglucin, p, 88).

Expt. 74. Destruction of the lignin element in wood. Take some paper which gives
the phloroglucin reaction for lignin strongly and cut it up into pieces about an inch
square. Then boil the paper in some 1 % sodium hydroxide solution for a short time.
After washing well, put it into a flask or large test-tube with a few c.c. of bromine-
water and allow it to stand for an hour or two. Then wash again and heat in a
2 % solution of sodium sulphite. Wash free from sulphite, dry and test with
alcoholic phloroglucin solution and strong hydrochloric acid. No red colour, or very
little, will be produced. If a little red coloration is formed, the process should be
repeated until finally all the lignin reaction disappears,

v] . HYDROLYZING ENZYMES 69

Pecto -cellulose. The non-cellulose constituents in this case belong
to the class of pectic substances which have already been considered
(see p. 63). Such celluloses occur in the cell-walls of the tissues of
many fleshy roots, stems and fruits.

Adipo- and cuto- celluloses. These products are found in the walls
of corky and cuticularized tissues. Their chemical composition is
obscure but they appear to contain substances of a fatty or wax-like
nature.

Hemi-celluloses. These are not strictly celluloses since they afe built
up of mannans, galactans and pentosans on lines which have already been
considered (see pp. 59 and 60). They frequently occur united with each
other, for instance as galacto-, gluco- and fructomannan, galactoaraban,
galactoxylan, etc. They are found in the cell-walls of the tissues of
many seeds.

THE SYNTHESIS AND INTER-RELATIONSHIPS OF CARBOHYDRATES

IN THE PLANT.

Now that the properties and characteristics of various carbohydrates
have been dealt with, their synthesis and their relationships, one to
another, may be considered.

In the previous chapter it has been shown how the plant synthesizes
a sugar from carbon dioxide and water by virtue of the chemical energy
obtained from transformation of radiant energy by means of chlorophyll.
When this sugar reaches a certain concentration in the cell, in the
majority of plants, starch is synthesized from it by condensation with
elimination of water. The starch is thus the first visible product of
assimilation and is temporarily " stored " in an insoluble form during the
day, when photosynthesis is active. During the night photosynthesis
ceases but the sugar is still translocated from the leaf, as it was in fact
during the day ; thus, since the supply ceases, the concentration in the
cell falls, and the " stored " starch is then hydrolyzed again into sugar,
and the process continues until the leaf is either starch-free, or contains
considerably less starch. During the next day, the starch formation is
repeated and so forth. The process of hydrolysis of starch is carried out
by the enzyme, diastase, with the formation of dextrin and maltose. In
all probability this same enzyme controls the synthesis of starch.

On the other hand, it has been shown that many plants do not form
starch at all in their leaves but only sugar. Examples are the adult

70 CARBOHYDRATES AND THEIR [GH.

Mangold plant (Beta vulgaris} and many Monocotyledons (Allium,
Scilla).

As to the question of which sugars are present in the leaf, there is
only evidence from accurate work on a few plants. Careful investiga-
tions have been made of the sugars in leaves of the Mangold (Beta
vulgaris) (Davis, Daish and Sawyer, 15), Garden Nasturtium (Tropaeo-
lum majus) (Brown and Morris, 5), the Snowdrop (Oalanthus nivalis)
(Parkin, 24), the Potato (Solanum tuberosum) (Davis and Sawyer, 17)
and the Vine (Vitis vinifera). The general conclusions drawn from
these investigations are that sucrose, glucose, and laevulose are always
present in leaves : that maltose results from the hydrolysis of starch,
b.eing absent from leaves which do not form starch. Maltose is not pre-
sent in appreciable quantity even in starch-producing leaves because it is
rapidly hydrolyzed into glucose by maltase. (In such cases where it has
been detected it has been due to diastase action during the drying of
leaves before extraction.) Other leaf carbohydrates are the pentoses
which have been found in a good many species examined and may be
widely distributed ; the pentosans, their condensation products, also occur
as well as dextrin (Potato).

The next question to be considered is what sugar is first synthesized
in the leaf. Is it glucose, laevulose, sucrose or maltose ? It is known
that the enzymes, invertase and maltase, are commonly present in leaves
and that these enzymes respectively control the hydrolysis, of cane-
sugar into glucose and laevulose, and of maltose into glucose. It is also
possible that they respectively control the synthesis of sucrose and
maltose. Laevulose, likewise, as may be supposed, can be obtained from
glucose. Thus all the sugars can be readily converted one into another, but
to ascertain which is the first product of synthesis is not an easy problem.

In addition to the above-mentioned work on the nature of the sugars
present in leaves, a good deal of careful analysis has been made as to the
proportions in which the sugars occur relatively to each other during
stated periods of time, with a view to answering the question as to
which is the first-formed sugar. There are two possibilities : one, that
it is sucrose and that it is hydrolyzed into glucose and fructose: the
other, that it is glucose, from which fructose is derived, and the two are
then synthesized to form sucrose.

Opinion is divided on this point and there is not at present sufficient
experimental evidence to decide the matter. The majority of investi-
gators regard sucrose as the first-formed sugar, and suggest that it is
inverted into hexoses for purposes of translocation, since the smaller

v] HYDROLYZING ENZYMES 71

molecules would diffuse faster. There is experimental evidence that
there is an increase in hexoses in the conducting tissues. Others favour
the view that glucose is the first-formed sugar, and bring forward
evidence to this effect. There is however no reason why hexoses should
not be formed first and then converted into cane-sugar and temporarily
stored as such, being again reinverted into hexoses for translocation.
Nor is there any reason for supposing that the first-formed sugar is
always the same in every plant.

There appears to be very little doubt that maltose is formed in
the hydrolysis of starch, and also that starch is a temporary reserve
material in the leaves, but whether formed direct from sucrose or from
hexoses cannot be stated.

There is some evidence in favour of the view that glucose is more
readily used in respiration than laevulose, for under circumstances when
neither can be increased, the glucose tends to disappear.

From the leaf the various sugars are translocated to other organs of
the plant, e.g. root, stem, flower, fruit and seed. In some cases starch
is synthesized from the sugars and " stored " in roots, tubers, tuberous
stems, fruits and seeds. In other cases the sugars themselves may be
" stored," as, for instance, in the root of the Beet (Beta vulgaris), or they
may have a biological significance, as in sweet fruits. It must also be
borne in mind that sugars are employed throughout the plant in re-
spiration and in the synthesis of more complex substances, i.e. cellulose,
gums, pentosans, mucilage, aromatic substances, fats and to a certain
extent proteins: in fact they or their precursors constitute the basis
from which all organic compounds are synthesized.

The following experiments can be performed with either the Garden
Beet or the Mangold Wurzel, both of which are varieties of Beta vulgaris,
the Common Beetroot. The sugars in the leaves and petioles of the
Mangold have been investigated (Davis, Daish and Sawyer, 15) and
sucrose, laevulose and glucose have been found. Starch is absent in
the adult plant and also maltose. The opinion is held that sucrose is
the first- formed sugar of photosynthesis and that this is hydrolyzed for
translocation on account of the greater rate of diffusion of the smaller
molecules of glucose and laevulose. These are again synthesized in the
root to form sucrose where the latter is stored, and hexoses are almost
absent from this organ. Though the facts concerning the distribution
of the sugars stated above are reliable, it is not certain that the deduc-
tions are permissible. The leaf contains the enzymes, invertase, maltase
and diastase (Robertson, Irvine and Dobson, 25).

72 CARBOHYDRATES AND THEIR [CH.

In connexion with the occurrence of various sugars in leaves it is
of interest to note that glucose, fructose and mannose can pass over
into one another in alkaline aqueous solution. This has been explained
by their conversion into the enolic (unsaturated) form common to all
three hexoses :

CHO CHO CH2(OH) CH(OH)

I

HCOH

HOCH

CO

C'OH

1

I

1

I

HOCH

HOCH

HOCH

HOCH

I

I

1

|

HCOH

HCOH

HCOH

HCOH

HCOH

HCOH

HCOH

HCOH

I

1

I

|

CH2(OH)

CH2(OH)

CH2(OH)

CH2(OH)

Glucose

Mannose

Fructose

Enolic form

Expt. 75. To show the presence of both hexoses and sucrose in the leaf (Davis,
Daish and Sawyer, 15). Take about 5 gms. of fresh leaf of either the Beet or Mangold.
(Leaves of the Garden Nasturtium (Tropaeolum majus) and Wild Chervil (Chaero-
phyllum sylvestre] may also be used.) Tear them into small pieces and drop them
into boiling 90-98 % alcohol in a flask on a water-bath. In this way the enzymes of the
leaf are killed, and no changes will occur in the carbohydrates present. After boiling for
a short time, the alcohol is filtered off and the extraction repeated. Evaporate the
filtrate to dryness in an evaporating dish on a water-bath. The filtrate will contain
chlorophyll and various pigments, sugars, glucosides, aromatic compounds and other
substances according to the plant used. Then add about 20 c.c. of water and at in-
tervals a few drops of basic lead acetate until it ceases to form a precipitate. By this
means all hexoses combined with aromatic substances as glucosides (see/p. 142) are
precipitated as insoluble lead salts. The precipitate is filtered oft' and the lead in the
fitrate removed by sodium carbonate, avoiding excess. Filter again and the filtrate
will contain the sugars. Boil the latter and add Fehling's solution drop by drop till
reduction ceases. Filter off the copper oxide and then boil the solution with dilute
sulphuric acid for a few minutes and make neutral to phenolphthalein. Reduction
will occur on adding more Fehling and boiling, owing to the inversion of the cane-
sugar present.

Expt. 76. To show the presence of hexoses in the leaf by means of the formation of
glucosazone. Leaves of Beta, Chaerophyllum sylvestre, or Tropaeolum may be used.
Extract as in the previous experiment and precipitate the glucosides with the
minimal amount of basic lead acetate. Test for osazone in the filtrate as in
Expt. 41 (d\

Expt. 77. To obtain starch from green leaves. Weigh out 25 gms. of leaflets of the
Pea (Pisum sativum}. The leaves should have been picked in the evening after a
sunny day, and it does not matter if the cut leaves are left overnight. Dip the leaf-
lets for a moment into boiling water, remove excess of water and drop them into
200 c.c. of 96-98 o/0 alcohol and boil till the chlorophyll is extracted : then filter.

v] HYDROLYZING ENZYMES 73

Take the residue of leaves and pound (but not finely) in a mortar and then wash
thoroughly with distilled water. Filter through muslin and press free from water
(this process extracts most of the protein). Boil the residue with 100 c.c. of water and
filter. To the filtrate add iodine. At first the colour may disappear owing to the
presence of protein in solution in addition to the starch. When more iodine is added
a deep blue coloration is formed.

PLANT ENZYMES WHICH HYDROLYZE CARBOHYDRATES.

Diastase. In the plant starch may be regarded as a reserve product.
It is synthesized from sugar, and may be again hydrolyzed into sugar.
It can be shown experimentally that starch is converted into glucose by
boiling with acids, but in the plant the hydrolysis of starch is catalyzed
by the enzyme, diastase. Although the reaction is doubtless of con-
siderable complexity, it may, broadly speaking, be represented as

follows :

(C6H1005)H+H20 — ^ (CeH^O^ + C^H^On
Dextrin Maltose

Thus the final products under these conditions are dextrin and the
disaccharide, maltose ; and not glucose.

It is reasonable to assume that cells which contain starch also either
contain, or are capable of producing, diastase. But the amount of
diastase present, or at any rate capable of being extracted, varies in
different tissues. Diastase, like most enzymes, is soluble in water. In
many cases, however, a water-extract from fresh crushed tissues in which
diastase occurs, will not contain any appreciable amount of enzyme.
This is sometimes due to the fact that the protoplasm does not readily
yield up the enzyme until it has been killed. If the tissues are dried at
a moderate temperature (30-40° C.) both the powdered leaves them-
selves and a water extract are fairly rich in diastase ; or, if the living
tissues are macerated and extracted with water to which chloroform
has been added, the cells die more rapidly and yield up the enzyme to
the solvent. From such a water extract, a crude precipitate containing
the enzyme may be obtained by addition of alcohol. For obtaining the
maximum results with diastatic activity in leaves, a water extract
should be made after they have been killed, either by drying, or by the
action of toluol or chloroform.

It has been shown (Brown and Morris, 5) that in leaves which con-
tain tannin, the presence of the latter largely inhibits the action of the
enzyme and may be the cause, in such cases, of an entire lack of activity
in the extract.

74 CARBOHYDRATES AND THEIR [CH.

The diastatic activity of leaves appears to vary largely in different
genera and species. The subject has been investigated (Brown and
Morris, 5) and a list of their relative activities has been drawn up as
follows.

[The numbers represent the amount of maltose, expressed in grams, which 10
gms. of air-dried leaf will produce from soluble-starch (starch treated with dilute
hydrochloric acid) by hydrolysis in 48 hrs. at 30° C.]

Pisum sativum 240*30 Heliaiithus animus 3*94

Phaseolus multiflorus 110-49 H. tuberosus 3*78

Lathyrus odoratus 100*37 Funkia sinensis 5*91

L. pratensis 34-79 Allium Cepa 3*76

Trifoliuui pratense 89*66 Hemerocallis fulva 2 '07

T. ochroleucum 56*21 Populus sp 379

Viciasativa 79*55 Syringa vulgaris 2*53

V. hirsuta 53*23 Cotyledon Umbilicus 4*61

Lotus corriiculatus 19*48 Humulus Lupulus 2*01-9*60

Lupinus sp 3*51 Hymenophyllum demissum . . . 4*20

Grass with Clover 27*92 Hydrocharis Morsus-ranae ... 0*267

Tropaeolum majus 3*68-9*64

From the above table it is seen that the leaves of genera of the
Leguminosae are apparently very rich in diastase. Whether this is so,
or whether in other plants the diastatic activity is inhibited by other
substances, has not yet been ascertained. As mentioned above, tannins
inhibit the action of diastase, and hence leaves rich in tannin, e.g. Hop
(Humulus), cannot be expected to yield good results.

The tissues of germinating barley (Hordeum vulgare) also contain
large quantities of diastase, and this material can be used to demon-
strate the solubility, isolation and activity of the enzyme.

The action on starch of diastase from the leaf of the Common Pea
(Pisum sativum) and from germinating barley grains has already been
demonstrated [see Expt. 57 (a) and (b)] in connexion with dextrin. The
following experiments have special reference to the enzyme.

Expt. 78. To demonstrate the activity of diastase from germinating barley. Grind
2-3 gms. of barley grains in a coffee-mill. Boil the product with 100 c.c. of water
and filter, first through fine muslin if necessary, then through "filter-paper. A starch
" solution " will be obtained.

Pound up 2-3 gms. of germinated barley grains in a mortar and extract the mass
with 50 c.c. of water. Filter, and take two equal portions in two test-tubes. Boil one
tube. To both tubes add an equal quantity of the starch solution prepared above.
Place the tubes in a beaker of water at 38-40° C. From time to time withdraw a
drop from each tube with a pipette and test with iodine solution on a white tile. The
starch in the unboiled tube will gradually give the dextrin reactions (see p. 57); that
in the boiled tube will remain unchanged.

v] HYDROLYZING ENZYMES 75

This simple method may also be adopted for showing the diastatic activity of
leaves. Instead of germinating barley, a few leaflets of the Pea (Pisum sativum] or
Clover (Trifolium pratense} should be pounded up in a mortar and extracted with
50 c.c. of water and filtered.

Expt. 79. To show that leaf-diastase w still active after drying the leaves at
temperatures not higher than 38° C. Take 10 gms. of fresh Pea leaves and dry by
spreading them in the sun. Then powder arid finally dry in an incubator at 38° C.
Make up 500 c.c. of a 1 % solution of starch (see Expt. 55). To this add the dry leaf
powder together with a few drops of toluol and keep in an incubator at 38° C. Test
the solution with iodine from time to time and note the hydrolysis of the starch.

Expt. 80. To show that the action of diastase is impaired by contact of the enzyme
with alcohol. Pound up 10 gms. of fresh Pea leaves, add 100 c.c. of water, a few drops
of toluol and allow the mixture to stand for 12 hrs. Filter off the extract, and
add at least an equal bulk of 96-98% alcohol. A white precipitate is produced
which, among other substances, contains crude diastase. Filter, and wash the
precipitate with a little water into 500 c.c. of a 1 % starch solution. Add a few drops
of toluol, plug with cotton-wool and put in an incubator. Test with iodine from
time to time. It will be found that the hydrolysis takes place much more slowly
than in the previous experiments.

Expt. 81. To show that the action of diastase is inhibited by tannic acid. Take
about O'5-l grn. of dried powdered Pea leaf. Let it stand for about 12 hrs. in 50 c.c.
of water containing a few drops of toluol. Filter off, and to equal amounts of the
filtrate in two small flasks add about 10 c.c. of a 1 % starch solution. Add also
10 c.c. of a 0-5 % tannic acid solution to one test-tube. Put both tubes into an
incubator. Test with iodine solution after a few hours. It will be found that the
tannic acid has inhibited the action of the diastase.

Maltase. This enzyme hydrolyzes maltose into two molecules of

glucose :

CigHfflOn -I- H2O = 2C6H12O6.

Investigations upon maltase have, until recently, produced rather
contradictory results, but later work (Davis, 12: Daish, 13, 14) has led to
more satisfactory conclusions. The latter show that maltase is most
probably present in all plants in which hydrolysis of starch occurs. It
has been detected in leaves of the Nasturtium (Tropaeolum), the Potato
(Solanum), the Dahlia, the Turnip (Brassica), the Sunflower (Heliantkus)
and the Mangold (Beta), and it is most probably widely distributed in
foliage leaves. Its detection is not easy for various reasons which are
as follows. It is not readily extracted from the tissues by water : it is
unstable, being easily destroyed by alcohol and chloroform. Its activity
is also limited or even destroyed at temperatures above 50° C. Hence
the extraction of maltase, by merely pounding up tissues with water,
does not yield good results : moreover, as an antiseptic, toluol must be

76 CARBOHYDRATES AND THEIR [CH.

used and not chloroform. Finally, if the enzyme is to be extracted from
dried material, this must not be heated at too high a temperature
previous to the extraction.

Maltase occurs in quantity in both germinated and ungerminated
seeds of cereals. If, in kilning, malt has not been heated at too high a
temperature, the maltase may not be destroyed, and, in such cases, malt
extract will contain both diastase and maltase. This would explain the
fact that glucose, instead of maltose, has sometimes been obtained by
the action of malt diastase on starch. In other cases, when a higher
temperature has been employed, the maltase will be destroyed. Maltase
itself, of course, does not act directly upon starch but only on maltose.
The use of chloroform, as an antiseptic, by some observers explains how
they came to overlook the presence of maltase, thus obtaining maltose,
and not glucose, as an end product in hydrolysis by malt extracts. The
optimum temperature for the maltase reaction is 39° C.

The presence of maltase in leaves is not readily shown for the
following reasons. Since maltase is destroyed by alcohol, the prepara-
tion of a crude precipitate of the enzyme by precipitating a water
extract of the leaves is not satisfactory. If the water extract is added
directly to maltose, and incubated, hydrolysis may be demonstrated by
determining the reducing power of the sugars formed. A control
experiment must, however, be made by incubating the water extract
alone, and subsequently determining the reducing power of any sugars
present.

Invertase. This enzyme hydrolyzes cane-sugar into one molecule
of glucose and one molecule of laevulose :

C12H22On + H20 = C6H1206 + C6H120G.

Invertase is probably very widely distributrcHn plants. Its presence
has been demonstrated in the leaves and stem, though not in the root,
of the Beet (Beta vulgaris) (Robertson, Irvine and Dobson, 25). Also in
the leaves of a number of other plants (Kastle and Clark, 20). Its de-
tection, by its action on sucrose, is not easy on account of the presence
of other enzymes and reducing sugars in leaf extracts.

The absence of invertase from the root of the Beet raises a difficulty
as to how the cane-sugar is synthesized from the hexoses supplied from
the leaves (see p. 71). Some observers (Robertson, Irvine and Dobson,
25) incline to the view that cane-sugar is synthesized in the stems and
travels as such to the roots. Others (Davis, Daish and Sawyer, 15)
maintain that the cane-sugar is synthesized in the root, even though
invertase is absent.

v] HYDROLYZING ENZYMES 77

REFERENCES

BOOKS

1. Abderhalden, E. Biochemisches Handlexikon, n. Berlin, 1911.

2. Armstrong, E. P. The Simple Carbohydrates and the Glucosides.
London, 1919. 3rd ed.

3. Atkins, W. R. G. Some Recent Researches in Plant Physiology. London,
1916.

4. Mackenzie, J. E. The Sugars and their Simple Derivatives. London,
1913.

PAPERS

5. Brown, H. T., and Morris, G. H. A Contribution to the Chemistry
and Physiology of Foliage Leaves. J. Chem. Soc., 1893, Vol. 63, pp. 604-677.

6. Czapek, F. LTeber die sogenannten Ligninreactionen des Holzes. Zs.
physiol. Chem., 1899, Vol. 27, pp. 141-166.

7. Davis, W. A., and Daish, A. J. A Study of the Methods of Estimation
of Carbohydrates, especially in Plant-extracts. A new Method for the Estimation
of Maltose in Presence of other Sugars. J. Agric. Sci., 1913, Vol. 5, pp. 437-468.

8. Davis, W. A., and Daish, A. J. Methods of estimating Carbohydrates. II.
The Estimation of Starch in Plant Material. The Use of Taka- Diastase. J. Agric.
Sri., 1914, Vol. 6, pp. 152-168.

9. Daish, A. J. Methods of Estimation of Carbohydrates. III. The Cupric
Reducing Power of the Pentoses — Xylose and Arabinose. J. Agric. Sri., 1914, Vol. 6,
pp. 255-262.

10. Davis, W. A., and Sawyer, G. C. The Estimation of Carbohydrates.
IV. The Presence of Free Pentoses in Plant Extracts and the Influence of other
Sugars on their Estimation. J. Agric. Sri., 1914, Vol. 6, pp. 406-412.

11. Davis, W. A. The Hydrolysis of Maltose by Hydrochloric Acid under
the Herzfeld Conditions of Inversion. A Reply to A. J. Kluyver. J. Agric. Sri.,
1914, Vol. 6, pp. 413-416.

12. Davis, W. A. The Distribution of Maltase in Plants. I. The Function
of Maltase in Starch Degradation and its Influence on the Amyloclastic Activity of
Plant Materials. Biochem. J., 1916, Vol. 10, pp. 31-48.

13. Daish, A. J. The Distribution of Maltase in Plants. II. The Presence
of Maltase in Foliage Leaves. Biochem. J., 1916, Vol. 10, pp. 49-55.

14. Daish, A. J. The Distribution of Maltase in Plants. III. The Presence
of Maltase in Germinated Barley. Biochem. J., -1916, Vol. 10, pp. 56-76.

15. Davis, W. A., Daish, A. J., and Sawyer, G. C. Studies of the Forma-
tion and Translocation of Carbohydrates in Plants. I. The Carbohydrates of the
Mangold Leaf. J. Agric. Sri., 1916, Vol. 7, pp. 255-326.

16. Davis, W. A. Studies of the Formation, etc. II. The Dextrose-Laevulose
Ratio in the Mangold. J. Agric. Sri., 1916, Vol. 7, pp. 327-351.

17. Davis, W. A., and Sawyer, G. C. Studies of the Formation, etc. III.
The Carbohydrates of the Leaf and Leaf Stalks of the Potato. The Mechanism of
the Degradation of Starch in the Leaf. J. Agric. Sri., 1916, Vol. 7, pp. 352-384.

78 CARBOHYDRATES AND ENZYMES [CH. v

18. Davis, W. A. The Estimation of Carbohydrates. V. The supposed
Precipitation of Reducing Sugars by Basic Lead Acetate. J. Agric. Sci., 1916, Vol. 8,
pp. 7-15.

19. Haynes, D. The Gelatinisation of Pectin in Solutions of the Alkalies and
the Alkaline Earths. Biochem. J., 1914, Vol. 8, pp. 553-583.

20. Kastle, J. H., and Clark, M. B. On the Occurrence of Invertase in
Plants. Amer. Chem. J., 1903, Vol. 30, pp. 421-427.

21. Neville, A. Linseed Mucilage. J. Agric. Sci.t 1913, Vol. 5, pp. 113-128.

22. Parkin, J. Contributions to our Knowledge of the Formation, Storage
and Depletion of Carbohydrates in Monocotyledons. Phil. Trans. R. Soc., B Vol. 191,
1899, pp. 35-79.

23. Parkin, J. On a Reserve Carbohydrate which produces Mannose, from
the Bulb of Lilium. Proc. Camb. Phil. Soc., 1900-1902, Vol. 11, pp. 139-142.

24. Parkin, J. The Carbohydrates of the Foliage Leaf of the Snowdrop
(Galanthus nivalis], and their Bearing on the First Sugar-of Photosynthesis. Biochem.
J., 1911, Vol. 6, pp. 1-47.

25* Robertson, R. A., Irvine, J. C., and Dobson, M. E. A Polarimetric
Study of the Sucroclastic Enzymes in Beta vulgaris. Biochem. J., 1909, Vol. 4,
pp. 258-273.

26. Salkowski, B. Ueber die Darstellung des Xylans. Zs. physiol. Chem.,
1901-2, Vol. 34, pp. 162-180.

27. Salkowski, B. Ueber das Verhalten des Arabans zu Fehling'scher
Losung. Zs. physiol. Chem., 1902, Vol. 35, pp. 240-245.

28. Schryver, S. B., and Haynes, D. The Pectic Substances of Plants.
Biochem. J., 1916, Vol. 10, pp. 539-547.

29. Schulze, E., Steiger, E., und Maxwell, W. Zur Chemie der Pflanzen-
zellmembranen. I. Abhandlung. Zs. physiol. Chem., 1890, Vol. 14, pp. 227-273.

CHAPTER VI

THE FATS AND LIPASES

A FAT may be defined as an ester or glyceride of a fatty acid. Just
as an inorganic salt, such as sodium chloride, is formed by the reaction
of hydrochloric acid with sodium hydroxide, so a fat is formed by the
reaction of the trihydric alcohol, glycerol, and a fatty acid.

The word fat is not a familiar one in botanical literature, the term
oil being more commonly used. It is generally met with in connexion
with the reserve products of seeds. The oils of seeds are, however, true
fats. The term oil may be misleading to some extent, because a fat
which is liquid at ordinary temperatures is usually spoken of as an oil,
and yet there are also many other substances, of widely differing chemi-
cal composition, which have the physical properties of oils, and which
are known as such.

Most of the vegetable fats are liquid at ordinary temperatures but
some are solids.

The best-known series of acids from which fats are formed is the
'series CnH^Oa of which formic acid is the first member. The other
members of the series which occur in fats are :

Acetic acid CH3COOH or C2H4O2
Butyric acid C3H7COOH or C4H3O2
Caproic acid C5HnCOOH or C6H12O,
Caprylic acid C7H15COOH or C8H16O2
Capric acid C9H19COOH or QoHjA
Laurie acid CnH^COOH or C12H24O2
Myristic acid C^H^COOH or CuK^A
Palmitic acid C15H31COOH or C16H3202
Stearic acid C17HMCOOH or
Arachidic acid C^H^COOH or
Behenic acid C^H^COOH or C^H^O,

Another series is the oleic or acrylic series CnH^.aOa of which the
members are :

Tiglic acid C5H8O2
Oleic acid C18HMOa
Elaidic acid C^H^O,
Iso-oleic acid C18H^O2
Erucic acid
Brassidic acid

80 THE FATS AND LIPASES [CH.

Of these, oleic acid (as glyceride) is the most widely distributed.
Yet other series are :

The linolic CnHan_4Oa

The linolenic CnH2n_6O2
The clupanodonic CnH2n_8O.2
The ricinoleic, CnH2n_203

The fat which occurs in an oil-containing seed is not composed of
the glyceride of one acid, but is a mixture of the glycerides of several,
or even a large number of different acids, often members from more
than one of the above series. Thus the fat of the fruit of the Coconut
(Cocos nuciferd) consists of a mixture of the glycerides of caproic, capry-
lic, capric, lauric, myristic, palmitic and oleic acids. Linseed oil from
the seeds of Linum usitatissimum again is a mixture of the glycerides
of palmitic, myristic, oleic, linolic, linolenic and isolinolenic acids. Simi-
lar mixtures are found in other fruits and seeds.

Since glycerol is a trihydric alcohol, it would be possible for one or
more of the three hydroxyls to react with the acid to form mono-, di- or
tri-glycerides. All these cases occur and, sometimes, one hydroxyl is
replaced by one acid, and another hydroxyl by a different acid.

When the distribution of fats among the flowering plants is con-
sidered, they are found to be more widely distributed than the botanist
is generally led to suppose.

The following is a list of some of the plants especially rich in fats
as reserve material in the fruits or seeds. It represents only a selection
of the better known genera, since many other plants have fatty seeds.
An approximate percentage of oil present in the fruit or seed is given.

Graminaceae : Maize (Zea Mays) 4 %.

Palmaceae : Oil Palm (Elaeis guinensis) 62 °/0 : Coconut Palm (Cocos
nucifera) 65 %.

Juglandaceae : Walnut (Juglans regia) 52 °/0.

Betulaceae : Hazel (Cory I us Avellana) 55 °/0.

Moraceae : Hemp (Cannabis sativa) 33 %.

Papaveraceae : Opium Poppy (Papaver somniferum) 47 %.

Cruciferae : Garden Cress (Lepidium sativum) 25 % : Black Mustard
(Sinapis nigra) 20°/0: White Mustard (Sinapis alba) 25°/0: Colza
(Brassica rapa var. oleifera) 33°/0: Rape (Brassica napus) 42°/0.

Rosaceae: Almond (Prunus Amygdalus) 42°/0: Peach (P. Persica)
35%: Cherry (P. Cerasus) 35%: Plum (P. domestica) 27%.

Linaceae : Flax (Linum usitatissimum) 20-40 %.

Euphorbiaceae : Castor-oil (Ricinus communis) 51 %.

vi] THE FATS AND LIPASES 81

Malvaceae: Cotton (Gossypium herbaceum) 24°/o-

Sterculiaceae : Cocoa (Theobroma Cacao) 54°/0.

Lecythidaceae : Brazil Nut (Bertholletia excelsa) 68 °/o-

Oleaceae: Olive (Olea europaea) 20-70%: Ash (Fraxinus excelsior)
27 o/0.

Rubiaceae : Coffee (Coffea arabica) 12°/0.

Cucurbitaceae : Pumpkin (Cucurbita Pepo) 41 °/0.

Compositae : Sunflower (Helianthus annuus) 38%.

The conclusion must not be drawn from the above list that the
seeds of the plants mentioned have exclusively fats as reserve materials.
In many cases fat may be the chief reserve product, but in others it
may be accompanied by either starch or protein or both.

Some of the best-known examples of fat-containing seeds which
yield " oils " of great importance in commerce, medicine, etc., are Ricinus
(castor oil), Brassica (colza oil), Gossypium (cotton-seed oil), Cocos
(coconut oil), Elaeis (palm oil), Olea (olive oil).

In the plant the fats are present as globules in the cells of the fat-
containing tissues.

Plant fats may vary from liquids, through soft solids, to wax-like
solids which generally have low melting-points. They float upon water
in which they are insoluble. They are soluble in ether, petrol ether,
benzene, chloroform, carbon tetrachloride, carbon bisulphide, etc. : some
are soluble in alcohol. With osmic acid fats give a black colour, and
they turn red with Alkanet pigment which they take into solution.

Expt. 82. Tests for fats. Weigh out 50 gins, .of Linseed (Linum usitatissimum)
and grind in a coffee-mill. Put the linseed meal into a flask, cover with ether, cork
and allow the mixture to stand for 2-12 hrs. Filter off the ether into a flask, fit
with a condenser and distil off the ether over an electric heater. (If a heater is
not available, distil from a water-bath of boiling water after the flame has been
turned out.) When the bulk of the ether is distilled off, pour the residue into an
evaporating dish on a water-bath and drive off the rest of the ether. With the residue
make the following tests in test-tubes :

(a) Try the solubilities of the oil in water, petrol ether, alcohol and chloroform.
It is insoluble in water and alcohol, but soluble in petrol ether and chloroform.

(6) Add a little 1 % solution of osmic acid. A black colour is formed. (This re-
action is employed for the detection of fat in histological sections.)

(c) Add to the oil a small piece of Alkanet (Anchusa officinalis) root, and warm
gently on a water-bath. The oil will be coloured red. Divide the oil into two portions
in test-tubes. To one add a little water, to the other alcohol. The coloured oil will
rise to the surface of the water in one case, and sink below the alcohol in the other.
The Alkanet pigment being insoluble in both water and alcohol, these liquids remain
uncoloured.

Keep some of the linseed oil for Expt. 83.
o. 6

82 THE FATS AND LIPASES [CH.

It is well known that the hydrocarbons of the unsaturated ethylene
series CnH^ will combine directly with the halogens, chlorine, bromine
and iodine to give additive compounds, thus :

ethylene bromide

The acids of this series also behave in the same way, and since many
plant fats contain members of the series, the fats will also combine with
the halogens.

Expt. 83. To show the presence of unsaturated groups in a fat. To a little of the
linseed extract add bromine water. Note the disappearance of the bromine and the
formation of a solid product.

One of the most important chemical reactions of fats is that known
as saponification. When a fat is heated with an alkaline hydroxide the
following reaction takes place :
C17H36CO-0— CH2

C17H3sCO • O— CH + 3KOH = 3C17H35COOK + CH2OH • CHOH • CH2OH

glycerol

C^H-tfCO'O— CH2
tristeariri

The potassium salt, potassium stearate, of the fatty acid, stearic
acid, is termed a soap. The ordinary soaps used for washing are mix-
tures of such alkali salts of the various fatty acids occurring in vegetable
and animal fats, and are manufactured on a large scale by saponifying
fats with alkali. The soaps are soluble in water, so that when a fat is
heated with a solution of caustic alkali, the final product is a solution of
soap, glycerol and excess of alkali. The soap is insoluble in saturated
salt (sodium chloride) solution, and when such a solution is added to
the saponified mixture, the soap separates out and rises to the
surface of the liquid. This process is known as " salting out." If the
saponified mixture is allowed to cool without salting out, it sets to
a jelly-like substance. When caustic potash is used for saponification
and the product is allowed to set, a " soft " soap is formed. Hard soaps
are prepared by using caustic soda and salting out.

The properties of soaps in solution are important. When a soap
goes into solution, hydrolysis takes place to a certain extent with the
formation of free fatty acid and free alkali. The free fatty acid then
forms an acid salt with the unhydrolyzed soap. This acid salt gives rise
to an opalescent solution and lowers the surface tension of the water
with the result that a lather is readily formed. *

The property of soaps of lowering surface tension is the reason for

vi] THE FATS AND LIPASES 83

their producing very stable emulsions when added to oil and water (see
chapter on colloids, p. 11).

Expt. 84. Hydrolysis of fat with alkali. Take 12 Brazil nuts, the seeds of Berthol-
letia (Lecythidaceae). Crack the seed coats and pound the kernels in a mortar. Put
the pounded nut in a flask, cover it with ether, and allow the mixture to stand for
2-12 hrs. Filter into a weighed or counterpoised flask and distil off the ether as in
Expt. 82. Weigh the oil roughly and add 4-5 times its weight of alcoholic caustic
soda (prepared by dissolving caustic soda in about twice its weight of water and
mixing the solution with twice its volume of alcohol). Heat on a water-bath until
no oil can be detected when a drop of the mixture is let fall into a beaker of water.
Then add saturated sodium chloride solution. The soaps will rise to the surface.
Allow the soaps to separate out for a time and then filter. Press the soap dry with
filter-paper, and test a portion to see that it will make a lather. Neutralize the
filtrate from the soap with hydrochloric acid and evaporate as nearly as possible to
dry ness on a water- bath. Extract the residue with alcohol and filter. Test the
filtrate for glycerol by means of the following tests :

(a) To a little of the solution add a few drops of copper sulphate solution and
then some sodium hydroxide. A blue solution is obtained owing to the fact that
glycerol prevents the precipitation of cupric hydroxide.

(6) Treat about 5 c.c. of a 0'5 °/0 solution of borax with sufficient of a 1 °/0 solu-
tion of phenolphthalein to produce a well-marked red colour. Add some of the
glycerol solution (which has first been made neutral by adding acid) drop by drop
until the red colour just disappears. Boil the solution : the colour returns. The re-
action is probably explained thus. Sodium borate is slightly hydrolyzed in solution
and boric acid, being a weak acid, is only feebly ionized, and therefore the solution
is alkaline. On adding glycerol, glyceroboric acid (which is a strong acid) is formed
and so the reaction changes to acid. On heating, the glyceroboric acid is hydrolyzed
to glycerol and boric acid, and the solution again becomes alkaline.

(c) Heat a drop or two with solid potassium hydrogen sulphate in a dry test-tube ;
the pungent odour of acrolein (acrylic aldehyde) should be noted :

C3H8O3 = C2H3 • CHO + 2H2O.

In addition to Brazil nuts, the following material can also be used :
endosperm of Coconut, ground linseed, almond kernels and shelled seeds
of the Castor-oil plant (Ricinus): about 50 gms. should be taken in
each case.

Expt. 85. Reactions of soaps, (a) Take some of the soap which has been filtered
oft' and shake up with water in a test-tube. A lather should be formed. (6) Make
a solution of a little of the soap in a test-tube and divide it into three parts. To each
add respectively a little barium chloride, calcium chloride and lead acetate solutions.
The insoluble barium, calcium and lead salts will be precipitated. (The curd which
is formed in the case of soap and hard water is the insoluble calcium salt.) Thirdly,
take the remainder of the soap and acidify it with dilute acid in an evaporating dish,
and warm a little on a water-bath. The soap is decomposed and the fatty acids are
set free and rise to the surface.

6—2

84 THE FATS AND UPASES [CH.

Expt. 86. Reactions of fatty adds, (a) Try the solubilities in ether and alcohol
of the acids from the previous experiment. They are soluble, (b) Shake an alcoholic
solution of the fatty acids with dilute bromine water. The colour of the bromine is
discharged owing to the bromine forming additive compounds with the unsaturated
acids.

The question of the metabolism of fats in the plant is a very com-
plicated one and has not yet been satisfactorily investigated. All plants
may have the power of synthesizing fats, and a great number, as we have
seen, contain large stores of these compounds in the tissues of the
embryo, or endosperm, or both. The point of interest is that of tracing
the processes by which these fats are synthesized, and are again hydro-
lyzed and decomposed. The products of decomposition may serve for
the synthesis of other more vital compounds as the embryo develops,
and before it is able to synthesize the initial carbohydrates, and to
absorb the salts requisite for general plant metabolism.

One fact seems fairly clear, namely that when fat-containing seeds
germinate, an enzyme is present in the tissues which has the power of
hydrolyzing fats with the formation of fatty acids and glycerol. Such
enzymes are termed lipases.

The lipase which has been most investigated is that which occurs in
the seeds of the Castor-oil plant (Ricinus communis). It has been shown
that if the germinating seeds are crushed and allowed to autolyze
(p. 19) in the presence of an antiseptic, the amount of fatty acid in-
creases, whereas in a control experiment in which the enzyme has been
destroyed by heat, no such increase takes place (Reynolds Green, 9, 10),
Investigation has shown the enzyme to be present also in the resting
seed, but in an inactive condition as a so-called zymogen (Armstrong,
4, 5, 6, 7). The zymogen is considered to be a salt and, after acidifica-
tion with weak acids, the salt is decomposed, and the enzyme becomes
active. After the preliminary treatment with acid, however, the enzyme
is most active in neutral solution. The effect of acid on the zymogen
may be demonstrated by autolyzing the crushed seed with a little
dilute acetic acid ; the increase of acidity will be found to be much
greater than in the case of a control experiment in which acid has not
been added.

It has not been found possible to extract the enzyme from the
resting seed. An active material can be obtained by digesting the
residue, after extraction of the fat, with dilute acetic acid and finally
washing with water. This material can then be used for testing the
hydrolytic power of the enzyme on various fats.

vi] THE FATS AND LIPASES 85

There is little doubt that lipase catalyzes the synthesis of fats as
well as the hydrolysis ; tlje reaction, in fact, has been carried out to a
certain extent in vitro.

Expt. 87. Demonstration of the existence of lipase in ungerminated Ricinus seeds.

A. Remove the testas from about two dozen Ricinus seeds and pound the kernels
up in a mortar. Into three small flasks (a), (6) and (c), put the following :

(a) 2 gins, of pounded seed + 10 c.c. of water.

(6) 2 gms. of pounded seed + 10 c.c. of water + 2 c.c. of N/10 acetic acid.

(c) 2 gms. of pounded seed + 10 c.c. of water + 2 c.c. of N/10 acetic acid, and
boil well.

Add a few drops of chloroform to all three flasks, plug them with cotton-wool,
and allow them to incubate for 12 hours at 37° C. Then add 2 c.c. of N/10 acetic
acid to flask (a), and 25 c.c. of alcohol to all three flasks. Titrate the fatty acids
present with N/10 alkali, using phenolphthalein as an indicator. A greater amount
of fat should be hydrolyzed in (6) than in («), and also slightly more in (a) than in
(c). The addition of alcohol checks the hydrolytic dissociation of the soap formed on
titration.

B. Pound up about 15 gms. of Ricinus seeds which have been freed from their
testas, and let the pounded mass stand with ether for 12 hrs. Then filter, wash with
ether and dry the residue. Weigh out three lots, of 2 gms. each, of the fat-free meal
and treat as follows :

(a) Grind up the 2 gms. of meal in a mortar with 16 c.c. of N/10 acetic acid
(i.e. 8 c.c. of acid to 1 gin. of meal), and let it stand for about 15 minutes. Then
wash well with water to free from acid, and transfer the residue to a small flask.
Add 5 c.c. of castor oil, 2 c.c. of water and a few drops of chloroform.

(6) Treat the 2 gms. of meal as in (a), but, after washing, and before transferring
to the flask, boil well with a little distilled water. Add 5 c.c. of oil, 2 c.c. of water
and a few drops of chloroform.

(c) Put the 2 gms. of meal into the flask without treatment and then add 5 c.c.
of oil, 2 c.c. of water and a few drops of chloroform.

Incubate all three flasks for 12 hours, and then titrate with N/10 caustic soda,
after addition of alcohol as in A. A certain amount of acetic acid is always retained
by the seed residue, and this is ascertained from the value for flask (6). Flask (c)
will act as the control.

Another question to be considered is the mode of synthesis in the
plant of the complex fatty acids which form the components of the fats.
No conclusive work has been done in this direction, but many investi-
gators have held the view that the fats arise from carbohydrates, notably
the sugars. In fact, it has been shown that in Paeonia and Ricinus, as
the seeds mature, carbohydrates disappear and fats are formed.

The sequence of events, however, in the synthesis of fatty acids
from sugars is very obscure. If we examine the formulae, respectively,

of a hexose :

CH2OH • CHOH • CHOH • CHOH • CHOH • CHO

86 THE FATS AND LIPASES [CH. vi

and a fatty acid, e.g. myristic acid :

H3C— CH2 • CH2 • CH2 • CH2 ' CH2 * CH2 • CH2 • CH2 • CH2 • CH2 ' CH2 • CH2 • COOH
it is seen that three main changes are concerned in the synthesis of
such a fatty acid from sugar, i.e. reduction of the hydroxyl groups of the
sugar, conversion of the aldehyde group into an acid group, and finally
the condensation or linking together of chains of carbon atoms. An
interesting fact in connexion with this point is that all naturally occur-
ring fatty acids have a straight, and not a branched, carbon chain. It
has been suggested (Smedley, etc., 11-13) that acetaldehyde and a
ketonic acid, pyruvic acid, may be formed from sugar. By condensation
of aldehyde and acid, another aldehyde is formed with two more carbon
atoms. By repetition of the process, with final reduction, fatty acids
with straight chains are produced.

REFERENCES
BOOKS

1. Abderhalden, B. Biochem isches Handlexikon, in. Berlin, 1911.

2. Leathes, J. B. The Fats. London, 1910.

3. Allen's Commercial Organic Analysis. Vol. 2. London, 1910.

PAPERS

4. Armstrong, H. E. Studies on Enzyme Action. Lipase. Proc. R. Soc.,

1905, B Vol. 76, pp. 606-608.

5. Armstrong, H. B., and Ormerod, B. Studies on Enzyme Action.
Lipase. II. Proc. R. Soc,, 1906, B Vol. 78, pp. 376-385.

6. Armstrong, H. E., and Gosney, H. W. Studies on Enzyme Action.
Lipase. III. Proc. R. Soc., 1913, B Vol. 86, pp. 586-600.

7. Armstrong, H. E., and Gosney, H. W. Studies on Enzyme Action.
Lipase. IV. The Correlation of Synthetic and Hydroly tic Activity. Proc. R. Soc.,
1915, B Vol. 88, pp. 176-189.

8. Miller, E. C. A Physiological Study of the Germination of Helianthus
annuus. Ann. Hot., 1910, Vol. 24, pp. 693-726. Ibid. 1912, Vol. 26, pp. 889-901.

9. Reynolds Green, J. On the Germination of the Seed of the Castor-oil
Plant (Ricinus communis). Proc. R. Soc., 1890, Vol. 48, pp. 370-392.

10. Reynolds Green, J., arid Jackson, J. Further Observations on the
Germination of the Seeds of the Castor-oil Plant (Ricinus communis). Proc. R. Soc.,

1906, B Vol. 77, pp. 69-85.

11. Smedley, I. The Biochemical Synthesis of Fatty Acids from Carbohydrate.
J. Physiol., 1912, Vol. 45, pp. xxv-xxvii.

12. Smedley, I., and Lubrzynska, E. The Biochemical Synthesis of the
Fatty Acids. Biochem. «/., 1913, Vol. 7, pp. 364-374.

13. Lubrzynska, E., and Smedley, I. The Condensation of Aromatic
Aldehydes with Pyruvic Acid. Biochem. «/., 1913, Vol. 7, pp. 375-379.

CHAPTER VII

AROMATIC COMPOUNDS AND OXIDIZING ENZYMES

THE aromatic compounds may be defined as substances containing
the benzene carbon ring or a similar ring. A very great number occur
among the higher plants but of these many are restricted in distribution,
and may only be found in a few genera or even in one genus : others,
on the other hand, are widely distributed. At present our knowledge of
the part they play in general plant metabolism is obscure.

The more widely distributed aromatic plant products may be grouped
as:

1. The phenols, and their derivatives.

2. The aromatic alcohols, aldehydes and acids (including the tan-
nins), and their derivatives.

3. The flavone, flavonol and xanthone pigments, known as the soluble
yellow colouring matters.

4. The anthocyan pigments, known as the soluble red, purple and
blue colouring matters.

In connexion with the aromatic compounds it should be noted that
many of them contain hydroxyl groups, and one or more of these groups
may be replaced by the glucose residue, C6HUO5 — , with elimination of
water and the formation of a glucoside, in the way already described
(see p. 48). The majority of such compounds are sometimes classed
together as a group — the glucosides — regardless of the special nature of
the substance to which the glucose is attached (this course has been
followed to some extent in Chapter IX with compounds, the chief
interest of which lies in their glucosidal nature). In treating of the
aromatic substances in the following pages, mention will be made when
they occur as glucosides, this combination being in these cases only a
subsidiary point in their structure.

The various groups of aromatic substances will now be considered
in detail.

PHENOLS.

There are three dihydric phenols, resorcinol, catechol and hydro-
quinone, but of these only the last is known to exist in the free state in
plants. The two former frequently occur as constituents of complex
plant products, and may be obtained on decomposition of such complexes
by fusion with strong alkali, etc.

88 AROMATIC COMPOUNDS AND [CH.

OH

• a ;

OH OH OH

Resorcinol Catechol Hydroquinone

Hydroquinone has been found in the free state in the leaves and
flowers of the Cranberry (Vaccinium Vitis-Idaea). As a glucoside,
known as arbutin, it occurs in many of the Ericaceae (see also p. 151).

Phloroglucin is the only member of the trihydroxy phenols found
uncombined in plants. It is very widely distributed in the combined
state in various complex substances (Waage, 27).

OH

Phloroglucin

AROMATIC ALCOHOLS AND ALDEHYDES.

The following are some of the better known compounds of this group:
Saligenin, or salicylic alcohol, in the form of the glucoside, salicin,
occurs in the bark of certain species of Willow (Salix), and in the
flower-buds of the Meadow-sweet (Spiraea Ulmaria). Salicin is hydro-
lyzed by an enzyme contained in the plant in which it occurs, into
saligenin and glucose (see also p. 152).

CH2OH

OH

Saligenin

Salicylic aldehyde occurs in species of Spiraea and other plants.

Coniferyl alcohol, as a glucoside, coniferin, is found in various
conifers and also in Asparagus (Asparagus officinalis). Coniferin is
hydrolyzed by dilute acids or by enzymes (emulsin) into coniferyl alcohol
and glucose (see also p. 151).

OCH.

OH

Coniferyl alcohol

vn] OXIDIZING ENZYMES 89

Coniferyl alcohol when oxidized yields the aldehyde, vanillin (so
much used for flavouring), which occurs in the fruits of the Orchid
( Vanilla planifolia).

AROMATIC ACIDS.

Of this group the following are some of the best known representa-
tives :

Salicylic acid is a monohydroxybenzoic acid. It occurs both in
the form of esters and in the free state in various plants.

Protocatechuic acid is a dihydroxybenzoic acid. It has been found
in the free state in a few plants, but is more widely distributed as a
constituent of many plant products. As will be shown later it forms
the basis of one of the series of tannins.

COOH COOH

OH

Salicylic acid Protocatechuic acid

Gallic acid is a trihydroxybenzoic acid :

COOH

OH

It occurs free in gall-nuts, in tea, wine, the bark of some trees and
in various other plants. It forms a constituent of many tannins. It is
a crystalline substance not very readily soluble in cold but more soluble
in hot water. In alkaline solution it rapidly absorbs oxygen from the
air and becomes brown in colour.

Expt. 88. The extraction and reactions of gallic acid. Take 100 gms. of tea, dry
in a steam oven and grind in a mortar. Put the powder into a flask and cover well
with ether. The preliminary drying and grinding can be omitted, but if carried out
will make the extraction more complete. After at least 24 hrs. filter off" the extract,
and either distil or evaporate off the ether. The ether will be coloured deep green
by the chlorophyll present in the dried leaves, and a green residue will be left. A.dd
about 20 c.c. of distilled water to the residue, heat to boiling and filter. Heating
is necessary because the gallic acid is only sparingly soluble in cold water. Keep the
residue for Expt. 91. With the filtrate make the following tests; for (a), (6) and (c)
dilute a few drops of the filtrate in a porcelain dish :

(a) Add a drop of ferric chloride solution. A blue-black coloration is given.

90 AROMATIC COMPOUNDS AND [CH.

(6) Add a drop or two of iodine solution. A transient red colour appears.

(c) Add a drop or two of lime water. A reddish or blue coloration will be given.

(d) To a few c.c. of the nitrate in a porcelain dish add a little lead acetate solu-
tion. A precipitate is formed which turns red on addition of caustic potash solution,
and dissolves to a red solution with excess of potash.

(e) To a few c.c. of the nitrate in a test-tube add a little potassium cyanide
solution. A pink colour appears, but disappears on standing. On shaking with air it
reappears.

(f) To a few c.c. of the nitrate in a test-tube add a few drops of 10 % gelatine
solution. No precipitate is formed.

(g) To a few c.c. of the filtrate in a test-tube add a little lead nitrate solution.
No precipitate is formed.

TANNINS.

This is a large group of substances, many of which are of complex
composition. They arise in the plant from simpler compounds, such as
protocatechuic, gallic and ellagic acids. Their formation takes place in
various ways, either by condensation, accompanied by elimination of
water, or by oxidation, or both ; there may also be condensation with other
aromatic complexes.

The tannins are widely distributed in the higher plants and, although
no very systematic investigation has been made, it is obvious that some
plants are rich in these substances, others poor, and others, again,
apparently entirely without them. The tannins generally occur in
solution in the cells of tissues of the root, stem, leaf, fruit, seed and
flowers: sometimes they are confined to special cells, tannin-sacs, but
after the death of the cell, the cell-walls of the dead tissue become
impregnated with the tannin. In tannin-producing plants, the tannin
is generally found throughout the plant, and it probably tends to
accumulate in permanent or dead tissues, such as the bark (dead cortex
and cork), woody tissue, underground stems, etc.

Tannins appear to be more frequent in woody than in herbaceous
plants, though in the latter they naturally only accumulate in the
persistent underground stems and root-stocks.- In annuals, also, tannins
seem to be more rare : this may be due to the fact that in a short-lived
plant, comparatively little tannin is formed and is not so readily detected
as in the tissues of a perennial.

In certain plants which are highly tannin-producing and are also
woody perennials, the bark becomes very rich in tannins. These barks
are consequently of considerable commercial importance for tanning of

vn] OXIDIZING ENZYMES 91

leather. As examples may be taken species of Ca^salpinia, Spanish
Chestnut (Castanea), Eucalyptus, Oak (Quercus), Mangrove (Rhizophora),
Sumac (Rhu$). Tannins also occur in quantity in galls, especially on
species of Quercus.

As a class, the tannins are non-crystalline and exist in the colloidal
state in solution. They have a bitter astringent taste. They have
certain properties and reactions in common, i.e. they precipitate gelatine
from solution, are themselves precipitated from solution by potassium
bichromate, and give either blue or green colorations with solutions of
iron salts. Many tannins occur as glucosides but this is by no means
always the case.

It is possible to classify the tannins into two groups according as
to whether they are complexes derived from protocatechuic acid or gallic-
acid :

1. The pyrogallol tannins. These give a dark blue colour with
ferric chloride solution, and no precipitate with bromine water.

2. The catechol tannins. These give a greenish-black colour with
iron salts, and a precipitate with bromine water.

Expt. 89. Reactions of tannins. Take three oak galls (the brown galls formed by
species of Cynips on the Common Oak) and pound them finely in a mortar. Boil up
the powder well with a small amount of water in an evaporating basin and let stand
for a short time. Then filter. The filtrate will contain tannin together with
impurities. Make the following tests with the extract :

(a) Put 2 c.c. of the tannin extract into a small evaporating dish, dilute with
water, and add a drop or two of ferric chloride solution. A deep blue-black colour is
produced.

(6) Put 2 or 3 drops of the tannin extract into a small evaporating dish, and
dilute with water : add a little dilute ammonia and then a few drops of a dilute
solution of potassium ferricyanide solution. A red coloration will appear.

(c) To 5 c.c. of the tannin solution in a test-tube add some strong potassium
dichromate solution. The tannin will be precipitated.

(d) To about 5 c.c. of the tannin extract in a test-tube add a little lead acetate
solution. The tannin will be precipitated.

(e) Melt a little of a 10 % solution of gelatine by warming gently and then pour
drop by drop into a test-tube half full of tannin extract. The gelatine will be
precipitated.

For the above tests, in addition to galls, the bark stripped from two to three year
old twigs of Quercus may also be used, and will give the same reactions. The bark
should be cut into small pieces for extraction.

It should be noted that although many tannins give the above
reactions, it does not necessarily follow that all tannins will give all the
reactions.

92 AROMATIC COMPOUNDS AND [CH.

Expt. 90. To demonstrate the existence of pyrogallol and catechol tannins. The
existence of a pyrogallol tanniii which gives a blue reaction with iron salts has
been illustrated in the last experiment on the Oak galls and the bark from Oak twigs.
The bark of the Sumac (Rhus Coriarid] and the fruit pericarp, leaves and bark of the
Sweet Chestnut (Castanea vulgaris} may be used as additional material for pyrogallol
tannins.

For an iron-greening tannin strip off the outer bark from two to three year old
twigs of the Horse Chestnut (Aesculus Hippocastanuni). Cut or tear the bark into
small pieces and boil well with a little water in an evaporating dish. Filter and test
the nitrate with ferric chloride solution as in Expt. 89. A green coloration will be
given. Iron-greening tannins may also be extracted from the bark of twigs of the
Walnut (Juglans regia] and of the Larch (Larix europaea}.

In the case of both classes of tannins, in addition to the ferric chloride reaction,
the tests of Expt. 89 (c) and (e) should also be made on the extracts, in order to
confirm the presence of tannin, since other substances, such as flavones, may give a '
green colour with iron salts (see p. 94).

Some of the individual tannins will now be considered.

Gallotannic (or tannic) acid is one of the most important of the
pyrogallol tannins. It occurs in Oak galls and Oak wood, in tea, in the
Sumac (Rhus Coriaria), etc. According to recent investigations (Fischer
and Freudenberg, 8) tannic acid may be regarded as a compound of one
molecule of glucose with five molecules of digallic acid in which five
hydroxyls of the sugar are esterified by five molecules of acid :
CH2(OX) ' CH (OX) • CH • CH (OX) • CH (OX) • CH (OX)

— o 1

t

where X= — CO -C6H2(OH)2 "O • CO • C6H2(OH)3

Tannic acid is an almost colourless amorphous substance. It has an
astringent taste, is soluble in water and alcohol, only slightly soluble in
ether, and insoluble in chloroform. It is decomposed, by boiling with
2 °/o hydrochloric acid, into gallic acid.

Expt. 91. Extraction and reactions of tannic (or gallotannic) acid. By a crude
method a solution of gallotannic acid can be obtained from tea. About 5 gms. of the
residue, after the extraction with ether in Expt. 88, is again extracted with ether
once or twice which will remove all but traces of gallic acid. Boil up the residue from
ether with a little water and filter. With the filtrate make the following tests which
differentiate between gallic and gallotannic acid :

(a) To about 10 c.c. add a little 10 % gelatine. The gelatine is precipitated.

(6) To a little of the filtrate add a few drops of lead nitrate solution. The tannic
acid is precipitated.

The remaining tests are given in common with gallic acid. If the extract is too
coloured, dilute with water.

(c) Dilute a few drops of the filtrate with water in a porcelain dish and add a
drop of ferric chloride solution. A blue-black colour is given.

vii] OXIDIZING ENZYMES 93

(d) Dilute a few drops of the filtrate with water in a porcelain dish and add a
drop or two of iodine solution. A transient red colour is formed.

(<e) To a little of the nitrate in a test-tube add a few drops of potassium cyanide
solution. A reddish-brown colour is formed which changes to brown but becomes
red again on shaking with air.

In addition to tannic acid, a great many other tannins are known,
but their constitution is obscure.

Expt, 92. To demonstrate that in tannin-containing plants the tannin may be
also present in the leaves. Take about two dozen leaves of the Common Oak
(Quercus Robur) and pound them in a mortar. Then boil the crushed mass in an
evaporating dish with a little water. Filter, and with the filtrate make the tests for
tannin. Leaves of other trees also may be used, e.g. the Wig Tree (Rhus Cotinus),
Sweet Chestnut (Castanea vulgaris).

Expt. 93. To demonstrate that tannins may be present in herbaceous as well as
u -oody plants. Extract some leaves, as in the last experiment, of Scarlet Geranium
(Pelargonium zonale) and test for tannin.

Expt. 94. To demonstrate that tannins may be present in petals and fruits, in
addition to other parts of the plant. Extract and test for tannins as in the last experi-
ment, using petals of Pelargonium zonale, Common Paeony (Paeonia ojficinalis) or
Hose (any garden variety), inflorescence of Flowering Currant (Ribes sanguineum\
flowers of Horse Chestnut (Aesculus Hippocastanum) or pericarp of Sweet Chestnut
(Castanea}.

THE FLAVONE AND FLAVONOL PIGMENTS.

These yellow colouring matters are very widely distributed in the
higher plants (Shibata, Nagai and Kishida, 26). They are derived from
the mother substances, flavone and flavonol, the latter only differing
from the former in having the hydrogen in the central 7-pyrone ring
substituted by hydroxyl :

Flavone

The naturally occurring pigments, however, have additional hydro-
gen atoms replaced by hydroxyl groups, that is they are hydroxy-
flavones and flavonols, and the various members differ among each other
in the number and position of these hydroxyl groups. Some of the
members are widely distributed, others less so. Quite often more than
one representative is present in a plant.

94 AROMATIC COMPOUNDS AND [CH.

The flavone and flavonol pigments are yellow crystalline substances,
and as members of a class they have similar properties. They occur in
the plant most frequently as glucosides, one or more of the hydroxyl
groups being replaced by glucose, or, sometimes, by some other hexose,
or pentose. In the condition of glucosides, they are much less coloured
than in the free state, and, being present in the cell -sap in very dilute
solution, they do not produce any colour effect, especially in tissues
containing chlorophyll. Occasionally they give a yellow colour to tissues,
as in the rather rare case of some yellow flowers (Antirrhinum) where
colour is due to soluble yellow pigment.

In the glucosidal state, the flavone and flavonol pigments are, as a
rule, readily soluble in water and alcohol, but not in ether. In the non-
glucosidal state they are, as a rule, readily soluble in alcohol, somewhat
soluble in ether, but soluble with difficulty in water.

The flavone and flavonol pigments can be easily detected in any
tissue by the fact that they give an intense yellow colour with alkalies
( Wheldale, 29). If plant tissues be held over ammonia vapour, they turn
bright yellow, showing the presence of flavone or flavonol pigments: the
colour disappears again on neutralization with acids. (The reaction is
especially well seen in tissues free from chlorophyll, such as white flowers.)
This reaction will be found to be almost universal, showing how wide is
their distribution. With iron salts, solutions of the pigments give green or
brown colorations. With lead, insoluble salts are formed. Several of
the members are powerful yellow dyes, and hence some plants in which
they occur, such as Ling (Erica cinerea), Dyer's Weld or Rocket (Reseda
luteola), have been used for dyeing purposes. The value of these colour-
ing matters as dyes has led to their chemical investigation, and as a
result the constitution, etc., of the hydroxy-flavones and flavonols is
well established.

Expt. 95. Demonstration of the presence of flavone or flavonol pigments in tissues
without chlorophyll. Take flowers of any of the undermentioned species and put
them in a flask with a few drops of ammonia. They will rapidly turn yellow owing
to the formation of the intensely yellow salt of the flavone or flavonol pigments
present in the cell-sap. If the flowers are next treated with acid the yellow colour
will disappear.

Also make an extract of some of the flowers with a little boiling water. Filter,
cool and add the following reagents :

(a) A little alkali. A yellow colour is produced.

(6) A little ferric chloride solution. Either a green or brown coloration is
produced.

(c) A little basic lead acetate solution. A yellow precipitate of the lead salt of
the flavone or flavonol pigment is formed.

vii] OXIDIZING ENZYMES 95

The flowers of the following species can be used : Snowdrop (Galanthus nivalis},
Narcissus (Narcissus poeticus), white variety of Lilac (Syringa vulgaris\ Hawthorn
(Crataegus OxyacantJia\ White Lily (Lilium candidum], white var. of Phlox, double
white Pink, white Stock (Matthiola) etc., etc., in fact almost any species with white
flowers* or a white variety.

Expt. 96. Demonstration of the presence of Jlavone or flavonol pigments in tissues
containing chlorophyll. Make a hot water extract of the leaves of any of the under-
mentioned species. Make with it the same tests as in the previous experiment.

The following plants may be used : Snowdrop (Galanthus nivalis\ Dock (Rumex
obtusifolius}, Goutweed (Aegopodium Podagraria), Dandelion (Taraxacum, ojficinale},
Violet ( Viola odoratd), Ribwort Plantain (Plantago lanceolata}^ Elder (Sambucus
nigra}.

The most important flavone pigments are apigenin, chrysin and
luteolin.

Apigenin has not yet been found to be widely distributed. Its
formula is :

OH

It occurs in the Parsley (Carum Petroselinum) (Perkin, 18) and in
the flowers of the ivory-white variety of Snapdragon (Antirrhinum
majus) (Wheldale and Bassett, 30).

Expt. 97. Extraction of apiin, the glucoside of apigenin, from the Parsley (Carum
Petroselinum). Take some Parsley leaves and boil in as little water as possible.
Filter off the extract and make the following tests for apigenin :

(a) Add alkali. A lemon yellow coloration is given.

(6) Add basic lead acetate solution. A lemon yellow precipitate is formed.

(c) Add ferric chloride solution. A brown colour is produced.

(d) Add ferrous sulphate solution. A reddish-brown colour is produced.

Apiin frequently separates out in a gelatinous condition from aqueous
and dilute alcoholic solutions.

Chrysin is a flavone occurring in the buds of various species of
Poplar (Populus). It has the formula :

96 AROMATIC COMPOUNDS AND [CH.

Luteolin does not appear to be widely distributed, though possibly
it occurs in many plants in which it has not yet been demonstrated. Its
formula is represented as :

OH

It occurs in the Dyer's Weld or Wild Mignonette (Reseda luteola)
(Perkin, 17), Dyer's Greenweed or Broom (Genista tinctoria) (Perkin, 23)
and in the yellow variety of flowers of the Snapdragon (A ntirrhinum
majus) (Wheldale and Bassett, 32). It has been much used as a yellow
dye: hence the names of the first two plants (Perkin and Horsfall, 20).

The most important flavonol pigments are quercetin, kaempferol,
myricetin and fisetin.

Quercetin is apparently one of the most widely distributed of the
whole group of yellow pigments, and has the formula :

It occurs, either free, or combined with various sugars (glucose,
rhamnose) as glucosides, in many plants, as for instance the following :
in the bark of species of Oak (Quercus), in berries of species of Buck-
thorn (Rhamnus), in flowers of Wallflower (Cheiranthus Cheiri), Hawthorn
(Crataegus Oxyacantha) (Perkin and Hummel, 22), Pansy ( Viola tricolor)
(Perkin, 19) and species of Narcissus : in leaves of Ling (Calluna erica)
(Perkin, 23), and the outer scale leaves of Onion bulbs (Perkin and
Hummel, 21).

Expt. 98. Preparation of a glucoside of quercetin from Narcissus flowers. The
species of Narcissus which can be used are N. Tazetta, N. incomparabilis, and any of
the common yellow trumpet varieties. Pound about 50 flowers in a mortar and then
extract in a flask with boiling alcohol. Filter off the alcoholic extract and evaporate
to dryness ; then add a little water and ether and transfer the whole to a separating
funnel. The ether takes up the plastid pigments, but at the plane of separation of the
liquids, the glucoside separates out as a crystalline deposit. This can be filtered off ;
with a dilute solution in alcohol make the following tests :

(«) Add a little alkali. The yellow colour is intensified, but the intensification
disappears on adding acid.

VII]

OXIDIZING ENZYMES

97

(6) Add a little lead acetate .solution. An orange precipitate of the lead salt is
formed.

(c) Add a little ferric chloride solution. A green coloration is produced.

(d) Heat some of the alcoholic solution on a water-bath, acidify with strong
hydrochloric acid and add zinc dust. A magenta colour is produced (see p. 106).

Expt. 99. Preparation of crude quercetin from Onion skins (Perkin, 21). Take
about 50 gms. of the brown outer skins of onions and boil with 900 c.c. of water for
an hour. Then filter and allow the filtrate to stand for 24 hrs. A brownish -yellow
deposit is formed which is crude quercetin. Filter this off and dissolve in 75%
alcohol and allow to evaporate slowly. Quercetin will be deposited. With a solution
in dilute alcohol make the same tests as in the last experiment.

Expt. 100. Preparation of a quercetin glucoside from Wallflower (Cheiranthus
Cheiri) flowers (Perkin and Hummel, 22). Take 20 gms. of petals of flowers of either
the brown or yellow variety and drop them into boiling alcohol in a flask. Filter and
evaporate the extract to dryness on a water-bath. Dissolve the residue in water and
add ether. In the case of the yellow variety the yellow plastid pigments are taken
up by the ether, and the quercetin glucoside partly crystallizes out from the water
as in Expt. 98. In the case of the brown variety both quercetin glucoside and
anthocyan pigment are present as well as plastid pigments. The two former go into
solution in the water and the glucoside in time crystallizes out. In either case the
glucoside can be filtered off and tested as in the previous experiment. A positive
result will be given in each case.

Kaempferol occurs in the flowers of a species of Larkspur (Delphi-
nium consolida) (Perkin and Wilkinson, 25) and Prunus (Perkin and
Phipps, 24) and in the leaves or flowers of several other plants. It has
the formula :

HO

Myricetin and fisetin are two other flavones which have been found
in species of Sumac (Rhus) and other plants. They have respectively
the formulae :

OH

HO

Myricetin

OH

o.

AROMATIC COMPOUNDS AND [CH.

THE ANTHOCYAN PIGMENTS.

These pigments are the substances to which practically all the blue,
purple and red colours of flowers, fruits, leaves and stems ,are due
(Wheldale, 3). They occur in solution in the cell-sap and are very
widely distributed, it being the exception to find a plant in which they
are not produced. As members of a group, they have similar properties,
but differ somewhat among themselves, the relationships between them
being much the same as those between the various flavone and flavonol
pigments. They occur in solution in the cell-sap but occasionally they
crystallize out in the cell. They are present in the plant in the form of
glucosides, and in this condition they are known as anthocyanins ; as
glucosides they are readily soluble in water and as a rule in alcohol
[except blue Columbine (Aquilegia), Cornflower (Centaurea Gyanus) and
some others] but are insoluble in ether and chloroform. The glucosides
are hydrolyzed by boiling with dilute acids, and the resulting products,
which are non-glucosidal, are termed anihocyanidins (Willstatter and
Everest, ^5). The latter, in the form of chlorides, are insoluble in ether,
but are generally soluble in water and alcohol. The anthocyanins can
be distinguished from the anthocyanidins in solution by the addition of
amyl alcohol after acidification with sulphuric acid. The anthocyanidins
pass over into the amyl alcohol, the anthocyanins do not. The antho-
cyanins and anthocyanidins themselves (with one exception) have not
yet been crystallized, but of both classes crystalline derivatives with
acids have been obtained (Willstatter and Everest, 35).

In considering the reactions of anthocyan pigments the difference
between those given by crude extracts and those of the isolated and
purified substances must be borne in mind. With acids the anthocyan
pigments give a red colour: with alkalies they give, as a rule, a blue or
violet colour when pure, but if flavone or flavonol pigments are present
(as may be the case in a crude extract) they give a green colour, due to
mixture of blue and yellow. In solution in neutral alcohol and water
many anthocyan pigments lose colour, and this is said to be due to the
conversion of the pigment into a colourless isomer which also gives a
yellow colour with alkalies (Willstatter and Everest, 35) ; hence even a
solution of a pure anthocyan pigment may give a green coloration with
alkali due to mixture of blue and yellow. The isomerization can be
prevented or lessened by addition of acids or of neutral salts which form
protective addition compounds with the pigment. With lead acetate

vn] OXIDIZING ENZYMES 99

anthocyan pigments give insoluble lead salts, blue if the pigment is
pure, or green, as in the case of alkalies, if it is mixed with flavone or
flavonol pigments, or the colourless isomer.

When anthocyan pigments are treated with nascent hydrogen, the
colour disappears but returns again on exposure to air. It is not known
what reaction takes place.

Expt. 101. The reactions of anthocyanins and anthocyanidins. Extract petals of
the plants mentioned below with boiling alcohol in a flask. Note that the anthocyan
colour may disappear in the alcoholic extract. Filter off some of the alcoholic extract
and make the following tests (a) and (6) with it :

(a) Add a little acid and note the bright red colour.

(b) Add a little alkali and note the green colour.

The decolorized petals, after filtering off the extract, should be warmed with a
little water in an evaporating dish. The colour is brought back if pigment is still
retained by them.

Evaporate the remainder of the alcoholic extract to dryness and note that the
anthocyan colour returns. Dissolve the residue in water and continue the following
tests, taking a little of the solution in each case :

(c) Add ether and shake. The anthocyan pigment is not soluble in ether.

(d) Add acid. A bright red colour is produced.

(e} Add alkali. A bluish-green or green colour is produced which may pass to
yellow.

(/) Add basic or normal lead acetate solution. A bluish-green or green precipitate
is produced.

(g) Add a little sulphuric acid and then amyl alcohol and shake : the latter does
not take up any of the red colour, indicating that the pigment is in the anthocyanin
(glucosidal) state.

(h] Heat a little of the solution on a water-bath with dilute sulphuric acid and
then cool and add amyl alcohol. The colour will pass into the amyl alcohol, indicating
that the pigment is now in the anthocyanidin (non-glucosidal) state.

(i) Acidify a little of the solution with hydrochloric acid and add small quantities
of zinc dust. The colour disappears. Filter off the solution and note that the colour
rapidly returns again.

For the above reactions it is suggested that the following flowers be used as
material : magenta Snapdragon (Antirrhinum majus\ brown Wallflower (Cheiranthus
Cheiri\ crimson Paeony (Paeonia officinalis\ magenta "Cabbage" Rose, Violet
( Viola odorata), but the majority of coloured flowers will serve equally well.

Though the above represent the reactions and solubilities given by
the greater number of anthocyan pigments, it will be found that all are
not alike in these respects.

Expt. 102. Demonstration that anthocyanins may be insoluble in alcohol but soluble
in water. Extract petals of any of the species mentioned belosv with boiling alcohol
and note that they do not lose their colour. It will be found that the pigments are
either completely or largely insoluble in alcohol, but are soluble in water. Test the

7—2

100 AROMATIC COMPOUNDS AND - [GEL

water extract as in Expt. 101 (c)-(i). Also take equal quantities of the water extract
in two evaporating dishes. To one add sodium chloride. Note (as mentioned above,
see p. 98) that the colour fades less rapidly from the extract containing the salt. The
..flowers of the following species can be used ": blue Larkspur (Delphinium), Cornflower
(Centaurea Cyanus], blue Columbine (Aquilegia).

There is a small group of plants belonging to some allied natural
orders, of which the anthocyan pigments give chemical reactions still
more different from the general type already described, though they
nevertheless resemble each other. Such, for instance, are the pigments
of various genera of the Chenopodiaceae [Beet (Beta), Orache (Atriplex)^
Amarantaceae (Amaranthus and other genera), Phytolaccaceae (Phyto-
lacca) and Portulacaceae (Portulaca). These anthocyan pigments are
insoluble in alcohol but soluble in water : they give a violet colour with
acids, red to yellow with alkalies, and a red precipitate with basic lead
acetate.

Expt. 103. Reactions of the Beet-root (Beta vulgaris) pigment. Take some Beet-
root leaves, tear them into small pieces and put them into alcohol. Allow the leaves
to stand for some time and note that the chlorophyll is extracted but the red pigment
is insoluble. Then pour off" the alcohol and add water : the red pigment goes into
solution. Filter off the solution and make the following tests :

(a) Add acid. The pigment turns violet.

(b) Add alkali. The pigment becomes redder and finally turns yellow.

(c) Add basic lead acetate. A red precipitate is formed.

(d) Acidify with hydrochloric acid and add zinc dust. The colour disappears, but
on filtering off from the zinc it does not return again.

Anthocyan pigments may also occur in leaves, and this is veiy obvious
in red-leaved varieties of various species such as the Copper Beech, the
Red-leaved Hazel, etc.

Expt. 104. Extraction of anthocyan pigment from the Red-leaved Hazel. Extract
some leaves of the Blood Hazel (Corylus Avellana var. rubra) with alcohol. Filter off
and evaporate the solution to dryness. Add water. Pour a little of the crude mixture
in the dish into a test-tube and add ether. There will be a separation into a green
ethereal layer containing chlorophyll, and a lower water layer containing anthocyan
pigment. Filter the extract remaining in the dish and with the filtrate make the
tests already given in Expt. 101 (c)-(i).

The leaves of the Copper Beech (Fagus sylvatica var. purpurea) can also be used.

In many flowers, the cells of the corolla may contain yellow plastid
(see p. 39) in addition to anthocyan pigments. The colour of the petals
is in these cases the result of the combination of the two, and is usually
some shade of brown, crimson or orange-red, as in the brown-flowered
variety of Wallflower (CheirantJius Clieiri).

VIl]

OXIDIZING ENZYMES

Expt. 105. Demonstration of the presence of anthocyan and plastid pigments
together in petals (see also Expt. 100). Extract petals of the brown-flowered variety of
Wallflower with alcohol. Filter, and evaporate the extract to dryness. Take up
with water and add ether. Pour the mixture into a separating funnel. The plastid
pigment will pass into solution in the ether, and the anthocyan pigment will remain
in the water. Test the aqueous solution as in Expt. 101 (c)-(t).

The following may be used as material : ray florets of bronze or crimson Chrys-
anthemum, ray florets of Gaillardia,&ud orange-red flowers of Nasturtium (Tropaeolum
majus}.

Anthocyanins and anthocyanidins have been isolated from various
species. The pigments themselves with one exception have not been
obtained in the crystalline state, but crystalline compounds with acids
have been prepared both of the glucosidal and non-glucosidal forms.

All the pigments so far described appear to be derived from three
fundamental compounds, pelargonidin, cyanidin and delphinidin, of
which the chlorides are represented thus :

OH H

Pelargonidin chloride

Cyanidin chloride

OH

Delphinidiu chloride

It has been suggested, at least in the case of cyanidin, the pigment
of the Cornflower (Gentaurea Cyanus), that the pigment itself is a neutral
substance, purple in colour and of the following structure (Willstatter,
33, 36):

102'

COMPOUNDS AND

[CH.

Further, that the blue pigment of the flower is the potassium salt
of the purple, and the red acid salt, cyanidin chloride, depicted above, is
a so-called oxonium compound of the purple.

Pelargonidin, moreover, has been prepared synthetically (Willstatter
and Zechmeister, 45).

The above three pigments, either as glucosides or in the form of
methylated derivatives, are found in a number of plants which are listed
below (Willstatter, etc., 33-46). The sugar residues or methyl groups
may, of course, occupy different positions, thus giving rise to isomers :

Callistephin
Pelargonin

Pelargonidin.

Monoglucoside of pelargonidin
Diglucoside of pelargonidin

Flowers of Aster (Callistephus chinensis]

Flowers of Scarlet Geranium (Pelargoniu

zonale), pink var. of Cornflower (Centaure

Cyanus} and certain vars. of Dahlia

variabilis]

Cyanidin.

Asterin

Chrysanthemin
Idaein
Cyanin

Mekocyanin
Keracyanin
Peonin

Monoglucoside of cyanidin
Monoglucoside of cyanidin
Monogalactoside of cyanidin
Diglucoside of cyanidin

Diglucoside of cyanidin
Rhamnoglucoside of cyanidin
Diglucoside of peonidin (cyanidin
monoethyl ether)

Flowers of Aster (Callistephus chinensis)
Flowers of Chrysanthemum (C. indicum]
Fruit of Cranberry ( Vaccinium Vitis-Idaea
Flowers of Cornflower (Centaurea Cyanus

Rosa gallica and certain vars. of Dahl

(D. variabilis}

Flowers of Poppy (Papaver Rhoeas]
Fruit of Cherry (Prunus Cerasus)
Flowers of Paeony (Paeonia officinalis)

Delphinidin.

Violanin
Delphinin

Ampelopsin

Myrtillin

Althaein

Petunin

Malvin

Oenin

Rhamnoglucoside of delphinidin
Diglucoside of delphinidin -f

p-hydroxybenzoic acid
Monoglucoside of ampelopsidin

(delphinidin monomethyl ether)
Monogalactoside of myrtillidin

(delphinidin monomethyl ether)
Monoglucoside of myrtillidin

Diglucoside of petunidin (delphi-
nidin monomethyl ether)

Diglucoside of malvidin (delphi-
nidin dimethyl ether)

Monoglucoside of oenidin (delphi-
nidin dimethyl ether)

Flowers of Pansy ( Viola tricolor)

Flowers of Larkspur (Delphinium consolida

Fruit of Virginian Creeper (Ampelopsis quir
quefolia]

Fruit of Bilberry ( Vaccinium Myrtillus)

Flowers of deep purple var. of Hollyhoc
(Althaea rosea)

Flowers of Petunia (P. violacea)
Flowers of Mallow (Malva
Fruit of Grape ( Vitis mnifera)

vii] OXIDIZING ENZYMES 103

Of the methylated compounds, myrtillidin and oenidin may be re-
presented thus:

Cl

HO H Hd H

Myrtillidin Oenidin

Expt. 106. Preparation and reactions of pelargonin chloride. Extract the flowers
from two or three large bosses of the Scarlet Geranium (Pelargonium zonale) in a
flask with hot alcohol. Filter off and concentrate on a water-bath. Then pour the
hot concentrated solution into about half its volume of strong hydrochloric acid. On
cooling, a crystalline precipitate of pelargouin chloride separates out. Examine under
the microscope and note that it consists of sheaves and rosettes of needles. Filter
off the crystals, take up in water and make the following experiments with the
solution :

(a) Add alkali. A deep blue-violet eolour is produced.

(b) Take two equal quantities of solution in two evaporating dishes. To one add
as quickly as possible some solid sodium chloride. The colour in the solution without
salt will rapidly fade owing to the formation of the colourless isomer in neutral
solution : this change is prevented to a considerable extent in the solution containing
salt owing to the formation of an addition compound of the pelargonin with the
sodium chloride which prevents isomerization (see p. 98). To portions of the water
solution (without sodium chloride) which has lost its colour add respectively acid
and alkali. The red colour returns with acid owing to the formation of the red acid
oxonium salt : with alkali a greenish-yellow colour will be produced due to the
formation of the salt of the colourless isomer. If alkali is added to the portion of the
pigment solution containing the sodium chloride, it will be found that it still gives
a violet colour.

(c) Add sulphuric acid and arnyl alcohol. The alcohol does not take up the
colour. Add amyl alcohol after acidifying another portion of the solution with
sulphuric acid and heating on a water-bath. The alcohol now abstracts some of the
colour. This shows that the glucoside pelargonin exists in the first case, but is
decomposed into the non-glucosidal pelargonidin after heating with acid.

(d) Acidify with hydrochloric acid and add zinc dust : the colour disappears and
returns again after filtering.

Expt. 107. Preparation of the acetic acid salt of pelargonin. Make an alcoholic
extract of petals as in Expt. 106. Evaporate down and pour into glacial acetic acid
instead of hydrochloric acid. The crystals of the salt formed are smaller and more
purple in colour than those of the chloride.

Expt. 108. Preparation and reactions of crude peonin chloride. Extract the petals
of one or two flowers of the Crimson Paeony (Paeonia officinalis] with 95-98%
alcohol. Filter and evaporate nearly to dryness. Then add some methyl alcohol and

104 AROMATIC COMPOUNDS AND [CH.

pour into a little strong hydrochloric acid. Then add ether to the mixture in a
separating funnel. A crude precipitate of peonin chloride will separate out after a
time, which may be more or less crystalline. Filter off this precipitate, take up in
water and make the following experiments :

(a) Take two equal quantities of the solution in two evaporating dishes. To one
add solid sodium chloride as in Expt. 106 (6). Then neutralize both portions carefully
with very dilute sodium carbonate solution until the colour changes slightly to
purple. The colour will fade more rapidly in the solution without sodium chloride
on account of the formation of a colourless isomer, as in the case of pelargonin
chloride. The water solution after standing will give a green colour with alkali owing
to admixture with the yellow salt of the isomer.

(6) Add alkali. A deep blue colour is produced. A crude extract of the fresh
petals made as in Expt. 101 will give a green or bluish-green colour with alkali owing
to the presence of the accompanying flavone.

(c) Add amyl alcohol and sulphuric acid. No colour is taken up by alcohol. The
pigment is present as the glucoside peonin. Boil another portion with sulphuric
acid and add amyl alcohol. The pigment is partly hydrolyzed and the peonidin goes
into solution in the alcohol.

(d] Eeduce another portion with zinc dust and hydrochloric acid. The colour
returns after filtering.

In considering the anthocyan pigments, the question now arises —
What is the chemical significance of the various shades in the living
plant ? Apparently the same pigment may be present in two flowers
of totally different colours, as in the blue Cornflower and the magenta
Rosa gallica. It has been suggested that in such cases the pigment is
modified by other substances present in the cell-sap: thus it may be
present in one flower as a potassium salt, in another as an oxonium salt
of an organic acid, and in a third in the unaltered condition. But exactly
how these conditions are brought about is not clear. In one or two
cases, moreover, where there is a red or pink variety of a blue or purple
flower, the variety, when examined, has been found to contain a different
pigment and one less highly oxidized than that in the species itself.
The above phenomena are exemplified in the Cornflower (Centaur ea
Cyanus). The flowers of the blue type contain the potassium salt of
cyanin, the purple variety, cyanin itself, while those of the pink variety
contain pelargonin.

The mode of origin of anthocyan pigments in the plant is as yet
obscure. It has been suggested (Wheldale, 29) that they have an
intimate connexion with the flavone and flavonol pigments, which can
be seen at once by comparing the structural formula of quercetin with
that suggested for cyanidin :

VII]

OXIDIZING ENZYMES

105

Quercetin

All the anthocyan pigments so far isolated, however, have been
found to contain the flavonol, and not the flavone, nucleus.

Just as in the case of the flavone and flavonol pigments, some of
the anthocyan pigments are specific, while others, on the contrary, are
common to various genera and species. Also more than one anthocyan
pigment may be present in the same plant.

It will be pointed out later that small amounts of a substance iden-
tical with cyanidin are said to be formed by reduction of quercetin with
nascent hydrogen, but this does not necessarily prove that the formation
of anthocyan pigments in the plant takes place on the same lines. If
we compare the formulae for a number of anthocyan with flavone and
flavonol pigments, it is seen that they may be respectively arranged in
a series, each member of which differs from the next by the addition of
an atom of oxygen :

Luteolin, kaempferol and fisetin C15H10O6
Quercetin C15H10O7
Myricetin C15H10O8

Pelargonidin C15H10O5
Cyanidin C15H1006
Delphinidin C15H10O7

The relationship between these two classes of substances in the
plant can only be ascertained by discovering which flavone, flavonol and
anthocyan pigments are present together, and then to determine whether
the relationship is one of oxidation or reduction, a problem which has
not yet received adequate attention (Everest, 6).

A reaction which is of interest in connexion with the relationship
between the above two classes of pigments is that which takes place
when solutions of some flavone or flavonol pigments are treated with
nascent hydrogen. If an acid alcoholic solution of quercetin is treated
with zinc dust, magnesium ribbon or sodium amalgam, a brilliant magenta
or crimson solution is produced, and this solution gives a green colour
with alkalies (Combes, 5). The red substance thus produced has been
termed " artificial anthocyanin " or allocyanidin. The product is not a

106 AROMATIC COMPOUNDS AND [CH.

true anthocyan pigment but has, it is suggested, an open formation
(Willstatter, 36):

OH

It is said, however, to contain small quantities of a substance iden-
tical with natural cyanidin from the Cornflower (Willstatter, 36). The
fact that small quantities of a natural anthocyan pigment can be obtained
artificially from a hydroxyflavonol by reduction does not necessarily imply
that one class is derived from the other in the living plant.

From the above reaction of quercetin the result follows that when
many plant extracts [most plants (see p. 94) contain flavone or ttavonol
pigments] are treated with nascent hydrogen, artificial anthocyan pig-
ment is produced. Moreover, it seems probable that if the yellow
pigments acted upon are in the glucosidal state, and if the reduction
takes place in the cold, allocyanin (the glucoside of allocyanidin) is
formed and the product is not extracted from solution by amyl alcohol.
But if the flavone is non-glucosidal, or if the solution is boiled before or
after reduction, then allocyanidin (non-glucosidal) is formed and is
extracted by amyl alcohol.

Expt. 109. Formation of allocyanidin from, quercetin. Make an alcoholic solution
of a little of the glucoside of quercetin prepared from either Narcissus or Cheiranthus
(see Expts. 98 and 100). Acidify with a little strong hydrochloric acid and heat on a
water-bath. Add a little zinc dust from time to time. A brilliant magenta colour
due to allocyanidin is produced. To a little of this solution add some alkali : a green
colour is produced. If the alcohol and hydrochloric acid are evaporated off, and a
little water and sulphuric acid added, on shaking up with amyl alcohol, all the
allocyanidin passes into the arnyl alcohol. (The distribution of the allocyanidin in
the amyl alcohol is greater with aqueous sulphuric acid than with aqueous hydro-
chloric acid.)

Expt. 110. Formation of allocyanin from quercetin. Make a suspension of the
glucoside of quercetin from Cheiranthus or Narcissus (see Expts. 98 and 100) in about 2N
sulphuric acid, and then add zinc dust (or a drop of mercury about the size of a pea
and a little magnesium powder) in the cold. The red or magenta colour is gradually
developed, Divide the coloured solution into two parts in two test-tubes. Boil one
for 5-10 minutes. Then add amyl alcohol to each. In the unboiled test-tube the
amyl alcohol extracts no colour, since allocyanin is present. In the boiled test-tube
allocyanidin is taken up by the amyl alcohol as in Expt. 109.

vii] OXIDIZING ENZYMES 107

Expt. 111. Formation of allocyanin and allocyanidin from plant extracts. For this
purpose the yellow varieties "Primrose" or "Cloth of Gold" of the Wallflower
(Cheiranthus Cheiri) can be used. The flowers are pounded in a mortar, extracted
with cold water, the water extract acidified with sulphuric acid, and zinc dust (or
mercury and magnesium powder as above) added. A red coloration is slowly
developed. To some of the red solution add aruyl alcohol. The colour is not
abstracted (allocyanin). Boil another portion. The allocyanin is thus converted into
allocyanidin which is then taken up on addition of amyl alcohol.

THE OXIDIZING ENZYMES.

There is a certain group of enzymes of which we have most informa-
tion in their connexion with aromatic substances. These are the oxi-
dizing enzymes.

The presence of such enzymes in plants was long 'ago associated with
the following phenomena. If the expressed juices, or water extracts of
the tissues, of some plants are added to a solution of guaiacum gum, in
the presence of air, a deep blue colour is obtained in a short time. On
the other hand, expressed juices, or water extracts, of other plants added
to guaiacum solution produce no blue colour. On addition, however, of
a few drops of hydrogen peroxide, in the latter case, the blue colour
rapidly develops. Plants are said to contain an oxidase when the extracts
give the blue colour with guaiacum tincture alone. Those, of which the
extracts only bring about blueing on addition of hydrogen peroxide, are
said to contain a peroxidase (Chodat, 1).

Expt. 112. Demonstration of the presence of an oxidase. Take a portion of a fresh
Potato tuber and pound well in a mortar with a little water. Filter, and to the
extract, in a white evaporating dish, add a few drops of 1 % guaiacum solution in
alcohol. The guaiacum tincture will be found to give a blue colour at once with the
potato extract.

A control tube should be prepared by well boiling some of the water extract of
the tuber, cooling and then adding guaiacum. No colour is produced, owing to the "
fact that the enzyme has been destroyed by boiling. Other tissues which may be
used to demonstrate the above reactions are : root of beet and fruits of apple, pear
and plum.

Guaiacum gum is obtained from two West Indian species of Guaiacum trees,
G. officinale and G. sanctum, partly as a natural exudation and partly by means of
incisions. It gives a yellow solution with alcohol which contains guaiaconic acid,
and the latter, on oxidation, yields guaiacum blue. As far as possible, inner portions
of the resin lumps should be used, as the resin oxidizes in air, and then may give
unreliable results. It is best to make the tincture freshly before use, and, as a
precaution, to boil it on a water-bath with a little animal charcoal and filter.
Guaiacum gum tends to form peroxides on exposure to air, and these are removed by
the above treatment.

108 AROMATIC COMPOUNDS AND [CH.

Expt. 113. Demonstration of the presence of a peroxidase. Repeat the above
experiment, using scales from an Onion bulb. Add a few drops of hydrogen peroxide
solution. Green pea tissue may also be used. No blue colour is produced until after
the addition of hydrogen peroxide.

If a systematic examination is made of genera from various natural
orders with the guaiacum test, it will be found that the oxidase reaction
is shown by all, or nearly all, genera of some orders, for instance most Um-
belliferae, Labiatae, Boraginaceae and many Compositae. In other orders,
i.e. Cruciferae and Crassulaceae, the peroxidase reaction is predominant.
Moreover, the oxidase-containing genera show another phenomenon,
as follows. If the tissue extracts are left in air, they become dark-
coloured, generally brown or reddish-brown. If the plants themselves
are bruised or injured, they turn brown. The same effect may be pro-
duced very rapidly by the action of chloroform vapour. These phenomena,
the darkening of extracts and the discoloration, both on injury and in
chloroform, will be found to be absent from plants giving the peroxidase
reaction only.

Expt. 114. To show the distribution of oxidases and peroxidases in various plants,
and the correlation between the presence of oxidase and browning on injury or in
chloroform vapour. Take a selection of the plants given below, and in each case grind
up a portion of the plant in a mortar with a little water and filter. Divide the
nitrate into two parts in small porcelain dishes. Allow one part to stand in air, and
note the darkening in colour in cases where an oxidase is present. To the other add
a few drops of guaiacum. To extracts containing a peroxidase only, after 5-10
minutes, add in addition a few drops of hydrogen peroxide. Further, small pieces
of the plants to be tested should be placed in a corked flask containing a few drops
of chloroform, and the development of browning noted in the case of plants contain-
ing an oxidase. For demonstration of oxidases the following plants may be used :
Christmas Rose (Helleborus niger}. Dandelion (Taraxacum officinale), Forget-me-not
(Myosotis\ Hawthorn (Crataegus) and White Dead Nettle (Lamium album}. For
peroxidases : Arabis, Aubrietia, Buttercup (Ranunculus acris\ Pea (Pisum sativum),
Stock (Matthiola\ Wallflower (Cheiranthus Cheiri) and Violet ( Viola}.

Various hypotheses have been suggested in explanation of the re-
actions of oxidizing enzymes. The one most generally accepted (Chodat,
1) is that the oxidase consists of two components, a peroxide and a
peroxidase, The peroxide may be either hydrogen peroxide or some
organic peroxide. The peroxidase acts upon the peroxide, depriving it
of an atom of oxygen which is transferred, in an " active " condition, to
some substance (acceptor) which becomes oxidized. Guaiacum is the
acceptor in the above experiments. The reaction may be approximately

expressed thus :

2AO + O2 = 2AO2 (peroxide).
AO2 -I- peroxidase = AO + O.

VII]

OXIDIZING ENZYMES

109

The above conception would represent a system which is capable of
absorbing molecular oxygen and of transforming it into active oxygen.
In this way substances can be oxidized which would not be affected
by molecular oxygen to any extent.

Accordingly, on the basis of the above hypothesis, it is assumed that
an organic compound which can act as a peroxide is present in the
expressed plant juices or extracts which give the oxidase reaction. When
this organic peroxide is absent, hydrogen peroxide must be added to
complete the oxidase system.

The above definition of oxidases and peroxidases is based upon their
behaviour towards guaiacum. There are, however, a number of other
oxidizable substances, such as a-naphthol, benzidine, p-phenylenediamine,
pyrogallol, etc. :

NH,

HoN

OH NH2

a-Naphthol Benzidine p-Phenylenediamine

which are only oxidized by oxidases with the assistance of hydrogen
peroxide, the oxidase system being insufficient ; they are also of course
oxidized by the hydrogen-peroxide-peroxidase system. Such substances
(in the presence of hydrogen peroxide) are used as tests for oxidizing
enzymes.

Expt. 115. Additional tests for oxidizing enzymes. To a few c.c. of an extract of
Potato tuber (Expt. 112) in a small evaporating dish, add a few drops of the following
reagents, and subsequently a few drops of hydrogen peroxide :

(a) A 1 % solution of a-naphthol in 50 °/0 alcohol. A lilac colour is developed.

(6) A 1 o/o solution of benzidine in 50 % alcohol. A blue colour is developed.

(c) A 1 % solution of jo-phenylenediamine hydrochloride in water. A greenish
colour is developed.

The peroxidases, like other enzymes, can be extracted either with
water or dilute alcohol and precipitated from solution by strong alcohol.

Expt. 116. Preparation of peroxidase from Horse-radish (Cochlearia) roots. Mince
up the Horse-radish roots in a mincing machine. The product is allowed to stand for
24 hrs. to enable the glucoside, potassium myronate, to be hydrolyzed by the enzyme,
myrosin. Then extract with 80 % alcohol. The alcohol is decanted off, and the
residue pressed free from alcohol in a press. The residue is next extracted with 40 %
alcohol for 48 hrs., filtered and precipitated with 90% alcohol. The precipitate,
which contains the peroxidase, is filtered off. Dissolve up in water and make the
test for peroxidases (Expt. 113).

110 AROMATIC COMPOUNDS AND [CH.

The oxidation of pyrogallol, in the presence of a peroxidase and
hydrogen peroxide, has been used as a method for estimating the activity
of these enzymes. Solutions of known strength of pyrogallol and hydro-
gen peroxide are used, and to the mixture a solution of a known weight
of prepared peroxidase is added. An oxidation product, termed purpuro-
gallin, is formed. After a definite time, the reaction is stopped by
adding acid, and the purpurogallin extracted by ether. The ether
extract is colorimetrically compared with an extract containing a known
amount of purpurogallin (Willstatter and Stoll, 47).

Expt. 117. Outline of method for estimating peroxidase by formation of purpuro-
gallin. Make a solution of 0'5 gm. of pyrogallol in 200 c.c. of distilled water, and
add to it 1 c.c. of 5 % hydrogen peroxide. Then add about 5 c.c. of a solution of
Horse-radish peroxidase from Expt. 116. After 5 minutes add to half the mixture
25 c.c. of dilute sulphuric acid and extract the purpurogallin with ether in a
separating funnel. The purpurogallin will be extracted by the ether, giving a yellow
solution. Allow the other half of the mixture to stand. The colour will deepen, and
a reddish deposit of purpurogallin will be precipitated. Examine a little of the
deposit under the microscope. It will be found to consist of sheaves of crystals.

A solution of peroxidase from Atyssum leaves [Expt. 119 (&)] can also be used.

It has recently been shown (Wheldale Onslow, 11) that the per-
oxidase element (Expt. 118) of certain oxidases (fruit of Pear, tuber of
Potato) can directly oxidize the phenol catechol (see p. 88), as well
as other substances containing the catechol grouping, that is two
hydroxyl groups in the ortho position, e.g. protocatechuic acid and caffeic

acid:

COOH CH=CH'COOH

OH

OH OH OH

Catechol Protocatechuic acid Caffeic acid

A brown colour is produced, giving rise to some oxidation product
which will then act as a peroxide, the peroxidase being present, to form
the system (oxidase) which will blue guaiacum.

It appears probable that the above phenomenon, as illustrated in
the Pear and Potato, affords an explanation of the reaction of the oxidiz-
ing enzymes of all plants which give the oxidase reaction and turn brown
on injury. For such plants usually contain an aromatic substance
with the catechol grouping. On the death of the cell (which may be
brought about, either by injury, or treatment with chloroform vapour,
i.e. on autolysis, see p. 19) this aromatic substance is oxidized by the

vii] OXIDIZING ENZYMES 111

peroxidase with the formation of a brown colour (browning on injury)
and an oxidation product which then acts as an organic peroxide (as
postulated above) in the oxidase system.

Further, the peroxidases, of plants which give the oxidase reaction
and brown on injury, appear to be specific in their action on the
catechol grouping and will not oxidize other phenolic groupings, i.e.
those of resorcinol, hydroquinone, pyrogallol, etc. \^^

In plants (e.g. Alyssum) which only give the peroxidase reaction and
do not brown on injury, the above-mentioned aromatic compounds are
not present, nor do the peroxidases of such plants act on catechol.

It should, however, be borne in mind that in some plants the com-
ponents of the oxidase system may be present, but the reaction is masked
by the presence of an excess of tannin, sugars, etc.

Hence it appears that the oxidase reaction of plants (as detected by
guaiacum) is the outcome of post-mortem changes after the death of
the cell. It is possible, however, that the processes giving rise to it
may take place to some extent, though under control, in the living cell.

It has also been shown that in the Pear fruit and Potato tuber
(Wheldale Onslow, 11) it is possible by chemical methods to separate
the elements which go to make up the oxidase system, i.e. the peroxidase
and the aromatic substance. If the tissues are ground up rapidly with
alcohol, and filtered before oxidation can take place, and the process
repeated, the aromatic substance, which is soluble in alcohol, is washed
away, and a water extract of the residue gives only a peroxidase reaction.
From the alcoholic extract the aromatic substance can be precipitated
as a lead salt, the lead then removed as insoluble lead sulphate, and the
aromatic set free again in solution. If the peroxidase is then added to
the solution of the aromatic, the brown colour appears together with the
formation of the oxidation product (peroxide), and the mixture will give
the blue reaction of an oxidase with guaiacum.

Expt. 118. Resolution of the components of the oxidase in the Potato tuber. (A)
Separation of peroxidase. Cut a few thin slices from a peeled potato and put them
in a mortar which contains sufficient 96 % alcohol to prevent, as far as possible,
exposure to the air, and pound them thoroughly. Filter quickly on a filter-pump,
and repeat the process several times until a colourless powder, consisting of cell-
residues, starch, etc. is obtained. The enzymes (including the peroxidase) of the cells
are precipitated by the alcohol and remain in the cell -residue. Make a water extract
of the white powder and filter. To a portion of the filtrate add a few drops of
guaiacum tincture : no blue colour is given. Add further a few drops of dilute
hydrogen peroxide : a blue colour appears. (B) Separation of the aromatic substance.
Take about 500 gms. of freshly peeled potato tuber, cut it into thin slices and drop

112 AROMATIC COMPOUNDS AND [CH.

them as rapidly as possible into a flask containing 250 c.c. of boiling 96 % alcohol
on a water-bath. Continue boiling for 15 mins., and then filter. Evaporate off the
alcohol from the nitrate, take up the residue in a little water, warm and filter. To
the filtrate add concentrated lead acetate solution until a precipitate ceases to be
formed. Filter off the precipitate, which is pale yellow in colour, stir up in a little
water and add 10 % sulphuric acid drop by drop until the yellow colour is destroyed,
and the lead is converted into lead sulphate. Filter off the lead sulphate : the filtrate
contains the aromatic substance in solution. Neutralize the solution carefully with
1 % caustic soda and make the following tests with separate portions in small
evaporating dishes :

(a) Add a drop of ferric chloride solution : a deep green colour appears. Add
further a few drops of 1 % sodium carbonate solution. The green colour changes to
a bluish- and finally, a reddish-purple. This reaction is characteristic of aromatic
compounds containing the catechol grouping, i.e. two hydroxyl groups in the
ortho position (see p. 110).

(6) Add a little of the peroxidase solution prepared in (A). The mixture will
gradually turn brown owing to the oxidation of the aromatic by the peroxidase.

(c) To (6) add a few drops of guaiacum tincture. A blue colour is given owing to
the presence of the peroxide formed in (6), the oxidase system being now complete.

Expt. 119. Action of peroxidases on catechol. (a) The peroxidase of the Potato
tuber (or Pear fruit}. Make a 1°/0 solution of catechol in distilled water. To some
of this solution, in a small evaporating dish, add a little of the peroxidase solution
from Expt. 118 (A). Note that the catechol solution gradually turns brown. Add
further a few drops of guaiacum tincture. A blue colour appears. (6) Peroxidase of
Alyssum leaves. Pound up 2-3 Alyssum leaves in a mortar with some 96% alcohol,
and filter on a filter-pump. Repeat the process until the residue is practically
colourless. Extract the residue with a little distilled water and filter. Proceed as in
(a). No browning of catechol takes place and no blue colour is formed on the
subsequent addition of guaiacum.

For section (a) the following material may also be used : fruits of Apple and
Greengage, flowers of Horse Chestnut (Aesculus) and leaves of Pear, the method of
preparation in (6) being employed. For section (6) flowers of white Arabis may also
be used.

If in the preparation of the peroxidase from the Potato tuber, the
tissue is allowed to brown before extracting with alcohol, the cell-residue
is tinged with brown and, on extraction with water, the nitrate will give
an oxidase reaction with guaiacum. This is to be explained by the fact
that the peroxide has been adsorbed by the tissue residue. This pheno-
menon is probably the explanation of the preparation of some oxidases
called " laccases." Such enzymes have been obtained by the precipita-
tion with strong alcohol of the expressed juices (containing peroxide
since they were obtained by crushing the tissues) of plants which brown
on injury. The enzyme and other organic matter is precipitated and
carries with it the peroxide.

vii] OXIDIZING ENZYMES 113

The browning of plants on injury, as already mentioned, is very
wide-spread : more rarely, the same type of reaction may lead to the
formation of variously coloured products, other aromatic substances in
addition to the peroxide being in all. probability involved. The following
examples may be quoted. In some of the Higher Fungi, Boletus luridus
and B. cyanescens, when the pileus is broken across or injured, the tissue
rapidly turns a deep Prussian blue on exposure to the air. Also in the
Fungus, Russula nigricans, the colour changes from pink or reddish to
black. The latex of the tree, Rhus vernicifera, when exposed to air,
turns black and constitutes- the product, Japanese lacquer.

Analogous pigments formed after death have been observed in
Schenckia blumenaviana (Rubiaceae) where the whole plant turns bright
red in chloroform vapour, as well as in species of Jacobinia and Aloe.
Blue pigments are also formed after death in the Teasel (Dipsacus), in
the flowers of an Orchid (Phajus), in the Dog's Mercury (Mercurialis
perennis), annual Mercury (M. annua) and in the well-known indigo-
producing plants, the Woad (Isatis tinctoria) and the Indigo (Indigofera)
(Walther, 28) (see p. 152). It has not been shown, however, that oxidizing
enzymes are present in all the above cases.

There is a well-known oxidizing enzyme, tyrosinase, which is some-
what specific in its action in that it will only oxidize tyrosine and a few
other substances, such as js-cresol. It occurs in the tubers of the Dahlia
(Dahlia variabilis) and Potato (Solarium tuberosum), in grains of barley
(Hordeum) and wheat (Triticum) and in Papaver orientale. In the lower
plants it is found among the Basidiomycetes.

When tyrosinase acts upon tyrosine, a yellowish-pink colour is first
produced which rapidly darkens to red, brown and finally black. A
solution of p-cresol

can be used as a delicate test for the enzyme. If the enzyme is present,
a yellowish- or orange-red colour is formed.

Tyrosinase of the Potato tuber can be precipitated from a water
extract with absolute alcohol : or if the potato tissue is extracted with
cold 96 °/o alcohol, the enzyme is precipitated and remains in the
o. 8

114 AROMATIC COMPOUNDS AND [CH.

tissue residue, as does the peroxidase [Expt. 118 (A)], but the tyrosine is
almost entirely washed away.

Expt, 120. Demonstration of the presence of tyrosinase in the Potato. Take about
half a potato and proceed as in the preparation of peroxidase [see Expt. 118 (A)].
Koughly dry the powder left on the filter and then add about 100 c.c. of water and
allow to stand for 15 mins. Filter, and divide the filtrate into six portions a, 6, c, rf,
e and /. Make a suspension of a little tyrosine in water (tyrosine is only slightly
soluble in cold water).

To a add 5 c.c. of tyrosine suspension.

To b add 5 c.c. of tyrosine suspension and a few drops of hydrogen peroxide.

To c add 5 c.c. of tyrosine suspension and boil.

To d add a few drops of hydrogen peroxide only.

To e add some p-cresol.

To /nothing is added.

Plug all the tubes with cotton-wool, put in an incubator at 38° C. for 2-3 hrs.
Note that tubes a and 6 fairly rapidly turn red, then brown and finally black.
Tubes d and / may darken a little owing to the action of tyrosinase and peroxidase
on the traces of plant aromatics left in the tissue. Tube c remains unaltered. Tube
e gives an orange-red colour.

Another point which may be touched upon is the significance of the
oxidizing enzymes in plant metabolism. The facts as they present
themselves may be stated thus. On death or injury we discover a
system (oxidase) in some plants which can oxidize certain acceptors,
either artificially introduced, such as guaiacum, or other aromatic sub-
stances present in the plant itself.

The question is whether the oxidase system as described above
functions in metabolism for the purpose of oxidation and respiration in
the living plant. If it does, there are various suggestions as to how it
may act. For instance, it is possible that aromatic substances are oxi-
dized to pigments by the oxidase system, and the pigment is then
deoxidized by products of anaerobic respiration, hence constituting a
mechanism for this function. Such pigments have been termed " respi-
ration pigments" (Palladin, 12-16). It is suggested that they are not
present in any quantity in the living plant and their production is
controlled by oxidizing and reducing systems :

autoxidizable substance + molecular oxygen = peroxide

peroxide + peroxidase = active oxygen

aromatic substance (acceptor or chromogen) -f active oxygen =

pigment
pigment 4- reducing substance = chromogen.

The above hypothesis is only applicable to plants containing oxi-

vn] OXIDIZING ENZYMES 115

dases, so that some modification would be necessary to fit the case of
plants containing peroxidases only.

It should be mentioned that the aromatic substances which become
oxidized are frequently present in the plant as glucosides, that is, some
of the hydroxyl groups are replaced by sugar. Indoxyl, the precursor of
indigo, forms a good example, as it is present in the plant as a colourless
glucoside, indican (see p. 152). It is probably only after the hydrolysis
of the glucosides that the aromatic substances can become oxidized.

REFERENCES
BOOKS

1. Chodat, R. Darstellung von Oxydasen und Katalasen tierischer und pflanz-
licher Herkunft. Methoden ihrer Anwendiing. Handbuch der biochemischen
Arbeitsmethoden. E. Abderhalden, Berlin, 1910, Vol. 3 (1), pp. 42-74.

2. Perkin, A. G., and Everest, A. B. The Natural Organic Colouring
Matters. London, 1918.

3. Wheldale, M. The Anthocyanin Pigments of Plants. Cambridge, 1916.

PAPERS

4. Clark, B. D. The Nature and Function of the Plant Oxidases. Torreya,
1911, VoL 11, pp. 23-31, 55-61, 84-92, 101-110.

5. Combes, R. Sur la presence, dans des feuilles et dans des fleurs ne formant
pas d'anthocyane, de pigments jaunes pouvant e"tre transformed en anthocyane.
C. R. Acad. sci., 1914, Vol. 158, pp. 272-274.

6. Everest, A. B. The Production of Anthocyanins and Anthocyanidins.
Part III. Proc. R. Soc., 1918, B Vol. 90, pp. 251-265.

7. Ewart, A. J. A Comparative Study of Oxidation by Catalysts of Organic
and Inorganic Origin. Proc. R. Soc., 1915, B Vol. 88, pp. 284-320.

8. Fischer, E., und Freudenberg, K. Ueber das Tannin und die Synthese
ahnlicher Stoflfe. Ber. D. chem. Ges., 1912, Vol. 45, pp. 915-935.

9. Keeble, P., and Armstrong, E. F. The Role of Oxydases in the Forma-
tion of the Anthocyan Pigments of Plants. J. Genetics, 1913, Vol. 2, pp. 277-311.

10. Moore, B., and Whitley, B. The Properties and Classification of the
Oxidising Enzymes, and Analogies between Enzymic Activity and the Effects of
immune Bodies and Complements. Biochem. J., 1909, Vol. 4, pp. 136-167.

11. Onslow, M. Wheldale. Oxidising Enzymes. I. The Nature of the
" Peroxide " naturally associated with certain direct Oxidising Systems in Plants.
Biochem. J., 1919, Vol. 13, pp. 1-9.

12. Palladin, W. Das Blut der Pflanzen. Ber. D. bot. Ges., 1908, Vol. 26 a,
pp. 125-132.

13. Palladin, "W. Die Verbreitung der Atmungschromogene bei den Pflanzen.
Ber. D. bot. Ges., 1908, Vol. 26 a, pp. 378-389.

14. Palladin, W. Ueber die Bildung der Atmungschromogene in den Pflanzen
Ber. D. bot. Ges., 1908, Vol. 26 a, pp. 389-394.

8—2

116 AROMATIC COMPOUNDS AND [CH.

15. Palladia, W. Ueber Prochromogene der pflanzlichen Atmungschromo-
gene. Ber. D. bot. Ges., 1909, Vol. 27, pp. 101-106.

16. Palladin, W. Ueber das Wesen- der Pflanzenatmung. Biochem. Zs.y
1909, Vol. 18, pp. 151-206.

17. Perkin, A. G. Luteolin. Part I. J. Chem. Soc., 1896, Vol. 69, pp. 206-
212. Part II. Ibid., 1896, Vol. 69, pp. 799-803.

18. Perkin, A. G. Apiin and Apigenin. J. Chem. Soc., 1897, Vol. 71, pp. SOS-
SIS. Ibid., 1900, Vol. 77, pp. 416-423.

19. Perkin, A. G. Robinin, Violaquercetin, Myrticolorin and Osyritrin.
J. Chem. Soc., 1902, Vol. 81, pp. 473-480.

20. Perkin, A. G., and Horsfall, L. H. Luteolin. Part III. J. Chem. Soc.,
1900, Vol. 77, pp. 1314-1324.

21. Perkin, A. G., and Hummel, J. J. Occurrence of Quercetinin the Outer
Skins of the Bulb of the Onion. J. Chem. Soc., 1896, Vol. 69, pp. 1295-1298.

22. Perkin, A. G., and Hummel, J. J. The Colouring Matters occurring
in various British Plants. Part I. J. Chem. Soc., 1896, Vol. 69, pp. 1566-1572.

23. Perkin, A. G., and Newbury, P. G. The Colouring Matters contained
in Dyer's Brooin (Genista tinctoria) and Heather (Calluna vulgaris). J. Chem. Soc.,
1899, Vol. 75, pp. 830-839.

24. Perkin, A. G., and Phipps, S. Notes on some Natural Colouring
Matters. J. Chem. Soc., 1904, Vol. 85, pp. 56-64.

25. Perkin, A. G., and Wilkinson, B. J. Colouring Matter from the
Flowers of Delphinium Consolida. J. Chem. Soc., 1902, Vol. 81, pp. 585-591.

26. Shibata, K., Nagai, I., and Kishida, M. The Occurrence and Physio-
logical Significance of Flavone Derivatives in Plants. J. Biol. Chem., 1916, Vol. 28r
pp. 93-108.

27. Waage, T. Ueber das Vorkommen und die Kolle des Phloroglucins in
der Pflanze. Ber. D. bot. Ges., 1890, Vol. 8, pp. 250-292.

28. Waltner, O. Zur Frage der Indigobildung. Ber. D. bot. Ges., 1909,
Vol. 27, pp. 106-110.

29. Wheldale, M. On the Nature of Anthocyanin. Proc. Camb. Phil. Soc.>
1909, Vol. 15, pp. 137-168.

30. Wheldale, M., and Bassett, H. LI. The Flower Pigments of Antirrhi-
num majus. II. The Pale Yellow or Ivory Pigment. Biochem. J., 1913, Vol. 7,
pp. 441-444.

31. Wheldale, M., and Bassett, H. LI. The Flower Pigments of Antirrhi-
num majus. III. The Ked and Magenta Pigments. Biochem. J., 1914, Vol. 8,
pp. 204-208.

32. Wheldale, M., and Bassett, H. LI. The Chemical Interpretation of
some Mendelian Factors for Flower-Colour. Proc. R. Soc., 1914, B Vol. 87,
pp. 300-311.

33. Willstatter, R. Ueber die Farbstoffe der Bliiten und Fruchte. SitzBer.
Ale. Wiss., 1914, pp. 402-411.

34. Willstatter, R., und Bolton, B. K. Ueber den Farbstoff der Schar-
lachpelargonie. Liebigs Ann. Chem., 1915, Vol. 408, pp. 42-61.

35. Willstatter, R., und Everest, A. B. Ueber den Farbstofi' der Korn-
blume. Liebigs Ann. Chem., 1913, Vol. 401, pp. 189-232.

vii] OXIDIZING ENZYMES 117

36. Willstatter, R., und Mallison, H. Ueber die Verwandtschaft der
Anthocyane und Flavone. SitzBer. Ale. Wiss., 1914, pp. 769-777.

37. Willstatter, R., und Mallison, H. Ueber den Farbstoff der Preiselbeere.
Liebigs Ann. Chem., 1915, Vol. 408, pp. 15-41.

38. Willstatter, R., und Mallison, H. Ueber Variationen der Bliitenfarben.
Liebigs Ann. Chem., 1915, Vol. 408, pp. 147-162.

39. Willstatter, R., und Martin, K. Ueber den Farbstoff der Althaea rosea.
Liebigs Ann. Chem., 1915, Vol. 408, pp. 110-121.

40. Willstatter, R., und Mieg, W. Ueber ein Anthocyan des Rittersporns.
Liebigs Ann. Chem., 1915, Vol. 408, pp. 61-82.

41. Willstatter, R., und Mieg, W. Ueber den Farbstoff der wilden Halve,
Liebigs Ann. Chem., 1915, Vol. 408, pp. 122-135.

42. Willstatter, R., und Nolan, T. J. Ueber den Farbstoff der Rose.
Liebigs Ann. Chem., 1915, Vol. 408, pp. 1-14.

43. Willstatter, R., und Nolan, T. J. Ueber den Farbstoff der Paonie.
Liebigs Ann. Chem., 1915, Vol. 408, pp. 136-146.

44. Willstatter, R., und Weil, P. J. Untersuchungen iiber Anthocyane,
Liebigs Ann. Chem., 1916, Vol. 412, pp. 113-251.

45. Willstatter, R., und Zechmeister, L. Synthese des Pelargonidins.
SitzBer. Ak. Wiss., 1914, pp. 886-993.

46. Willstatter, R., und Zollinger, B. H. Ueber die Farbstoffe der Wein-
traube und der Heidelbeere. Liebigs Ann. Chem., 1915, Vol. 408, pp. 83-109.

47. Willstatter, R., und Stoll, A. Ueber Peroxydase. Liebigs Ann. Chem.,
1918, Vol. 416, pp. 21-64.

CHAPTER VIII

THE PROTEINS AND PROTEASES

No class of compounds is of more fundamental significance than the
proteins. The matrix of protoplasm largely consists of proteins in the
colloidal state, and, without doubt, they occur to some extent in the
same condition in the cell-sap. They are also found in the cell in the
solid state, in the form of either amorphous granules, termed aleurone,
or crystalline or semi-crystalline bodies, termed crystalloids. Both solid
forms constitute " reserve material" and are often found in seeds, tubers,
bulbs, buds and roots.

Plant proteins may be classified on the following plan :

1. The simple proteins.

(a) Albumins.
(6) Globulins.

(c) Prolamins (Gliadins).

(d) Glutelins.

2. Conjugated proteins.

(a) Nucleoproteins.

3. Derived proteins.

(a) Metaproteins.

(6) Proteoses (Albumoses).

(c) Peptones.

(d) Polypeptides.

Although they are present in every cell in all parts of plants, little,
however, is known of plant proteins, except of those in seeds, because
of the difficulties of obtaining them in sufficiently large quantities, and
of separating them from each other.

Proteins are in the colloidal state when in so-called solution, and are
unable to diffuse through parchment membranes. The proteoses and
peptones, however, which have simpler molecules, can diffuse through
such membranes.

The vegetable proteins are soluble in various solvents according to
the nature of the protein ; some are soluble in water, others in dilute
salt solutions, others, again, in dilute alkalies, and a few in dilute
alcohol. Vegetable albumins are coagulated from solution on boiling,

CH. vin] THE PROTEINS AND PROTEASES 119

but most of the globulins, unlike the corresponding animal products,
are only imperfectly coagulated on heating and some not at all. The
precipitate formed when coagulation is complete will not go into solution
again either in water, acid, alkali or salts. Alcohol precipitates the
proteins ; in the case of animal proteins, the precipitate becomes coagu-
lated and insoluble if allowed to remain in contact with the alcohol, but
this does not appear to be so with plant proteins.

In addition, certain neutral salts, the chlorides and sulphates of
sodium, magnesium and ammonium, have the property of precipitating
proteins (except peptones) from solution when added in sufficient quan-
tity. The protein is quite unchanged in precipitation and can be made
to go into solution again. The various proteins are precipitated by
different concentrations of these salt solutions (see p. 124).

The salts of calcium and barium and the heavy metals produce
insoluble precipitates with the proteins, and in this case the reaction is
irreversible.

In regard to chemical composition, the proteins contain the elements
carbon, hydrogen, nitrogen, oxygen and sulphur. There is every reason
to believe that the protein molecule is constituted of amino-acids con-
densed, with elimination of water, on the plan which may be depicted
as follows :

Ri Rii Riii

i I , I

NH2— CH— COIOH HiNH— CH— CO|OH HiNH— CH— COiOH HiNH—

—COiOH H-NH— CH^-COOH

•.

Conversely, when the proteins are acted upon by hydrolyzing en-
zymes, a series of hydrolytic products are formed which have smaller
molecules than the original proteins. They may be enumerated as :

1. Albumoses.

2. Peptones.

3. Amino-acids.

In the same way when proteins are boiled with acids, a number of
the amino-acids are obtained as an end-product.

The above amino-acids may be either aliphatic or aromatic, and they
are characterized by having one or more hydrogen atoms, other than
those in the carboxyl groups, replaced by the group — NH2. Thus they
are acids by virtue of the carboxyl groups, and bases by virtue of the
— NH2 groups: towards strong acids they act as bases, and towards

120 THE PROTEINS AND PROTEASES [CH.

strong bases as acids. The amino-acid, alanine, for instance, forms salts,
sodium amino-propionate with a base, and alanine hydrochloride with
an acid :

CH3— CH— COONa CH3— CH— COOH

I I

NH2 NH2'HCI

Substances behaving in this way have been termed "amphoteric"
electrolytes (see also p. 15).

In the proteins, which are formed by condensation, as explained above,
there are always some NH2 and COOH groups left uncombined. Hence
a protein must, in the same way, have the properties of both an acid
and a base.

The amino-acids which are obtained by the hydrolysis of plant pro-
teins may be classified as follows :

Aliphatic compounds.

Mono-carboxylic morio-amino acids :
Glycine or a-amino-acetic acid

CH2(NH2)*COOH
Alanine or a-amino-propionic acid

CH3-CH(NH2)'COOH
Valine or a-amino-iso-valeric acid

CH3

>CH -CH(NH2)- COOH
CH/

Leucine or a-amino-iso-caproic acid

CH3X

>CH • CH2 ' CH(NH9) • COOH
CH/

Iso-leucine or a-amino-/3-niethyl-£-ethyl-propionic acid

-CH(NH2) -COOH

Serine or a-amino-^-hydroxy-propionic acid

CH2OH-CH(NH2)'COOH

Dicarboxylic mono-amino acids :

Aspartic acid or a-amino-succinic acid

COOH • CH2 • CH(NH2) • COOH
Glutaminic acid or a-amino-glutaric acid

COOH •CH2-CH2-CH(NH2)-COOH

vin] THE PROTEINS AND PROTEASES 121

Mono-carboxylic di-amino acids:

Arginine or 8-guanidme-a-amiuo-valeric acid
NH2

HN=C— NH • CH2 ' CH2 • CH2 • CH(NH2) • COOH
Lysine or a-e-di-amino-caproic acid

CH2(NH2) • CH2 • CH2 • CH2 • CH(NH2) • COOH

Dicarboxylic di-amino acid :

Cystine (dicysteine) or di-/3-thio-a-amino-propionic acid
CH2— S— S— CH2

CH(NH2) CH(NH2)

I I

COOH COOH '

•Aro)natic compounds.
Mono-carboxylic mono-amino acids :

Phenyl-alanine or /3-phenyl-a-amino-propionic acid
C6H5 • CH2 • CH(NH2) ' COOH
Tyrosiue or p-hydroxy-phenyl-alanine «

OH • C6H4 ' CH2 • CH(NH2) • COOH

Heterocyclic compounds.

Proline or a-pyrrolidine-carboxylic acid

CH2 CH2

I ' I

CH9 CH-COOH

Histidine or ^-iminazole-alanine
CH

NH N

CH=C— CH2 • CH(NH2) ' COOH

Tryptophane or /3-indole-alanine C8H6N • CH2 • CH(NH2) • COOH

-C'CH2-CH(NH2) -COOH

CH

Different proteins are formed by various combinations of the above
acids and hence give different amounts on hydrolysis.

There are certain properties and chemical reactions by means of

122 THE PROTEINS AND PROTEASES [CH.

which proteins can be detected. These are illustrated in the following
experiment.

Eocpt. 121. Tests for proteins. Weigh out about 10 gms. of dried peas (Pisum)y
grind them in a coffee-mill and then add 100 c.c. of water to the ground mass.
Allow the mixture to stand for an hour. Filter, and make the following tests with
the filtrate (see p. 133).

(a) The xanthoproteic reaction. To a few c.c. of the protein solution in a test-tube
add about one-third of its volume of strong nitric acid. A white precipitate is
formed (except in the case of proteoses, peptones, etc.). On boiling, the precipitate
turns yellow, and may partly dissolve to give a yellow solution. Cool under the tap,
and add strong ammonia till the reaction is alkaline. The yellow colour becomes
orange. The precipitate is due to the fact that metaprotein (see p. 127) is formed
by the action of acid on albumins or globulins, and this metaprotein is insoluble in
strong acids. The yellow colour is the result of the formation of a nitre-compound
of some aromatic component of the protein, such as tyrosine, tryptophane and
phenylalanine.

(6) Millon's reaction. To a few c.c. of the protein solution add about half its
volume of Millon's reagent1. A white precipitate is formed. On warming, the preci-
pitate turns brick-red, or disappears and gives a red solution. The white precipitate is
due to the action of the mercuric nitrate on the proteins. The reaction is character-
istic of all aromatic substances which contain a hydroxyl group attached to the
benzene ring. The aromatic complex in the protein to which the reaction is due is
tyrosine.

(c) The glyoxylic reaction (Hopkins and Cole}. To about 2 c.c. of protein solution
add an equal amount of "reduced oxalic acid"2. Mix the solutions, and then add an
equal volume of concentrated sulphuric .acid, pouring it down the side of the tube.
A purple ring forms at the junction of the two liquids. If the liquids are mixed by
shaking the tube gently, the purple colour will spread throughout the solution. The
substance in the protein molecule to which the reaction is due is tryptophane. If
carbohydrates are present in the liquid to be tested, the colour is not good, owing to
blackening produced by the charring with the strong sulphuric acid.

(d) The biuret reaction. To a few c.c. of the protein solution add an excess of
sodium hydrate and a drop of a 1 % solution of copper sulphate. A violet or pink
colour is produced. The reaction is given by nearly all substances containing two
CONH groups attached to one another, to the same nitrogen atom, or to the same
carbon atom. The cause of the reaction with proteins is the presence of one or more

1 This reagent is made by dissolving 30 c.c. of mercury in 570 c.c. of concentrated
nitric acid and then adding twice its bulk of water. It contains mercurous and mercuric
nitrates, together with excess of nitric acid and a little nitrous acid.

2 Reduced oxalic acid is prepared as follows : (a) Treat 500 c.c. of a saturated solution
of oxalic acid with 40 gms. of 2°/0 sodium amalgam. When hydrogen ceases to be
evolved, the solution is filtered and diluted with twice its volume of distilled water. The
solution contains oxalic acid, sodium binoxalate and glyoxylic acid (COOH-CHO).
(6) Put 10 .gms. of powdered magnesium into a flask and just cover with distilled water.
Add slowly 250 c.c. of saturated oxalic acid, cooling under the tap. Filter off the insoluble
magnesium oxalate, acidify with acetic acid and dilute to a litre with distilled water.

vin] THE PROTEINS AND PROTEASES 123

groupings formed by the condensation of the carboxylic group of an amino-acid with
the amino group of another amino-acid (see p. 119).

(e) The sulphur reaction. Boil a few c.c. of the protein solution with some 40 °/0
sodium hydrate for two minutes, and then add a drop or two of lead acetate. The
solution turns black. This reaction is due to the formation of sodium sulphide by
the action of the strong alkali on the sulphur of the protein. On addition of the lead
salt, a black precipitate of lead sulphide is formed. The sulphur in the protein
molecule is mainly present as cystiue.

For the following reactions, a protein solution free from other impurities is
required. For this purpose take 40 gms. of ground peas, add to the meal about
200 c.c. 10 °/0 sodium chloride solution, and allow the mixture to stand, with occa-
sional stirring, for 3-12 hrs. (see p. 134). Then filter off the extract, first through
muslin, and, subsequently, through filter-paper. Put the extract to dialyze for
24 hrs. in a collodion dialyzer1 until the protein is well precipitated. (Toluol
should be added to the liquid in the dialyzer.) Then filter off the protein. Reserve
half, and dissolve the other half in about 50 c.c. of 5 % sodium nitrate solution.
With this solution (after reserving a portion for Expt. 123) make the following tests :

(/) Precipitation by alcohol. To a few c.c. in a test-tube, add excess of absolute
alcohol. The protein is precipitated.

(g) Precipitation by the heavy metals. Measure out a few c.c. of the protein
solution into three test-tubes, and add respectively a little (1) copper sulphate
solution, (2) lead acetate solution, (3) mercuric chloride solution : the protein is
precipitated in each case.

The following test cannot be demonstrated on the Pea protein, since carbo-
hydrates are absent in this case. It can, however, be demonstrated in later
experiments (see p. 130).

(h) Molisch's reaction. To a few c.c. of the protein solution add a few drops of a
1 % solution of a-naphthol in alcohol. Mix, and then run in an equal volume of
strong sulphuric acid down the side of the tube. A violet ring is formed at the
junction of the two liquids. The reaction signifies the existence in the protein of a
carbohydrate group which gives rise, on treatment with acid, to furfural. The latter
then condenses with a-naphthol to give a purple colour (see also Expts. 38, 44, 46).

(i) Precipitation by salts of alkaline earth metals. To a few c.c. of the protein
solution add a little barium chloride solution. A precipitate is formed on standing.

(/) Precipitation by neutral salts. Saturate a few c.c. of the protein solution
with finely powdered ammonium sulphate. The protein is precipitated or "salted out."

Since from a neutral salt solution the pea globulin is precipitated by acid
(see p. 125), the tests (k}-(ra) should be carried out with a solution of the protein in
dilute acid. Dissolve, therefore, the remainder of the solid pea globulin in about
40 c.c. of 10 °/0 acetic acid, filter, and make the following tests :

(k) Precipitation by tannic acid. Add a little tannic acid solution : the protein is
precipitated.

1 The collodion solution is made by adding 75 c.c. of ether to 3 gms. of well-dried
pyroxylin, allowing it to stand for 10-15 minutes and then adding 25 c.c. of absolute
alcohol. The dialyzers are prepared by coating the inside of a large test-tube with the
solution and then filling with water, after the film is sufficiently dried so as not to be
wrinkled by touching with the finger. The sac can then be withdrawn from the tube.

124 THE PROTEINS AND PROTEASES [CH.

(1} Precipitation by Esbach's solution1. Add a little Esbach's solution: the pro-
tein is precipitated.

(m) Precipitation by phosphotungstic acid. Add a little 2 % solution of phos-
photungstic acid in 5 % sulphuric acid : the protein is precipitated.

The substances used in the tests (&)-(ra) are termed " alkaloidal re-
agents" because they also cause precipitation of alkaloids (see Chap. x).

We are now in a position to deal with the different groups of pro-
teins in detail :

Albumins. Very few vegetable albumins have been investigated.
They can be best defined as proteins which are soluble in water and are
coagulated by heat. Animal albumins are distinguished by the fact
that they are not precipitated by saturating their neutral solutions with
sodium chloride or magnesium sulphate ; nor are they precipitated by
half-saturation with ammonium sulphate. This distinction cannot be
Applied to vegetable proteins, since some are precipitated by the above
treatment. It is often not easy to determine whether a plant protein is
an albumin, on account of the difficulty of removing traces of salts, acids
or bases which cause solubility, and also of separating the albumins from
the globulins with which they occur. Albumins are however probably
widely distributed in plant tissues.

The best-known albumins are :

Leucosin, which occurs in the seeds of Wheat (Triticum vulgare),
Rye (Secale cereale) and Barley (Hordeum vulgare).

Legumelin, which occurs in seeds of the Pea (Pisum sativum), Broad
Bean ( Vicia Faba), Vetch ( Vicia sativa), Lentil (Ervum Lens) and some
other Leguminous seeds.

Phaselin, which occurs in the Kidney-bean (Phaseolus vulgaris).

Ricin, which occurs in the Castor-oil Bean (Ricinus communis).

Expt. 122. Demonstration of the presence of an albumin (leucosiri) in wheat or
barley flour (see also Expts. 128 and 130). Weigh out lOgms. of wheat or barley flour,
add 100 c.c. of distilled water and allow to stand, with occasional stirring, for
2-6 hrs. Then filter off the solution. Slowly heat the solution to boiling, and note
that a precipitate of coagulated protein is formed.

Globulins. These may be defined as the proteins which are in-
soluble in water but soluble in dilute salt solutions, the concentration of
the salt solution necessary for complete solution (see p. 125) varying
with the salt or protein under consideration. It should be noted that,
in making wafer-extracts of plant tissues, it may happen that globulins

1 Esbach's solution is prepared by dissolving 10 gms. of picric acid and lOgms. of citric
acid in water and making the solution up to a litre.

vin] THE PROTEINS AND PROTEASES 125

pass into solution to some extent owing to the presence of inorganic
salts in the tissues themselves. This has also already been illustrated
in Expt. 121 in which an extract of the globulin of the Pea was obtained
by treating ground Pea seeds with distilled water only.

It is characteristic of animal globulins that they are precipitated by
saturation of their solutions with magnesium sulphate. Many of the
vegetable globulins cannot be precipitated by the above means, though
they are all, as far as tested, precipitated by sodium sulphate at 33° C.
Many also (like animal globulins) are precipitated by half-saturation
with ammonium sulphate, though others are not precipitated until their
solutions are nearly saturated with this salt [see Expt. 121 (j)].

Unlike animal globulins, vegetable globulins are, as a rule, only
imperfectly coagulated by heat, even on boiling.

Expt. 123. Demonstration of the coagulation of globulin. Heat a few c.c. of the
solution of dialyzed Pea globulin (from Expt. 121) in a test-tube. Note that the
protein is largely precipitated, but the solution does not become quite clear.

One very important characteristic of the vegetable globulins is the
ease with which a number of them can be obtained in crystalline form.
This result may be achieved by dialyzing a salt solution of the globulin.
The salt passes out through the membrane, and the protein is deposited
in the form of crystals. An alternative method is to dilute the saline
solution of globulin with water at 50-80° C. until a slight turbidity
appears. Then warm further until this goes into solution, and cool
gradually, when the protein will separate in crystals. The globulin,
edestin, from seeds of the Hemp (Cannabis sativa) crystallizes very
readily (see Expt. 133) and crystals can also be obtained of the globulins
from the seeds of the Brazil nut (Bertholletia excelsa) (see Expt. 136),
the Flax or Linseed (Linum usitatissimum) (see Expt. 135), the Oat
(Avena sativa) and the Castor-oil plant (Ricinus communis) (see Expt.
134) ; other globulins separate out on dialysis as spheroids, sometimes
mixed with crystals.

The solubilities of plant globulins are further complicated by the
fact that some of these substances form acid salts which have different
solubilities from the proteins themselves. Thus edestin is insoluble in
water, but soluble in either dilute salt solution or acid. In the presence
of acid it forms salts which are insoluble in dilute salt solutions. Thus
edestin in dilute acid solution is precipitated by a trace of salt, or in
dilute salt solution by a trace of acid (see Expt. 124). Legumin, on the
other hand, from the Pea and other Leguminosae is soluble in water in

126 THE PROTEINS AND PROTEASES [CH.

the free state : combined with a small amount of acid as a salt, it is
insoluble in water but soluble in neutral salt solution, that is, it has the
solubilities of a globulin (see p. 134).

Expt. 124. The formation of salts by edestin. Grind up 5 gms. of seeds of the
Hemp (Cannabis sativa) in a coffee-mill. Extract with 50 c.c. of warm (not above
60° C.) 10°/0 sodium chloride solution and filter. Add a drop of strong hydrochloric
acid to the filtrate. Edestin chloride, which is insoluble in salt solutions, is precipi-
tated. Filter and drain off all the liquid, and then suspend the precipitate in
distilled water. Add 1 or 2 drops of hydrochloric acid carefully and stir till most or
all of the precipitate goes into solution. Filter, and to the filtrate add a few drops
of saturated sodium chloride solution. The edestin acid salt is again precipitated.

The following is a list of the principal known globulins (Osborne, 2):

/Pea (Pisum sativum).

T • • j r Broad Bean ( Vicia Faba).

Legumm, in seeds of -{ Tr . . _. .

Vetch ( Vicia sativa).

V Lentil (Ervum Lens).

Vignin, in seeds of Cow Pea ( Vigna sinensis).

Glycinin, in seeds of Soy Bean (Glycine hispida).

( Kidney Bean (Phaseolus vulgaris).
Phaseolin (crystalline), in seeds of -j Adzuki Bean (P. radiatus).

[Lima Bean (P. lunatus).
Conglutin, in seeds of Lupin (Lupinus).

fPea (Pisum sativum).
Vicilin, in seeds of -j Broad Bean ( Vicia Faba).

[Lentil (Ervum Lens).
Corylin, in seeds of Hazel Nut ( Corylus Avellana).

/Almond (Prunus Amygdalus).

. . Peach (P. Persica).

Amandin, in seeds of <tn , „ 7 . . .

rlum (Jr. aomestica).

(Apricot (P. Armeniaca).

f European Walnut (Juglans regia).
Juglansin, in seeds of -j American Black Walnut (J. nigra).

[American Butter-nut (J. cinerea).
Excelsin (crystalline), in seeds of Brazil Nut (Bertholletia excelsd).

Edestin, in seeds of Hemp ( Can nabis sativa).

Avenalin, in seeds of Oat ( Avena sativa).

Castanin, in seeds of Sweet Chestnut (Castanea vul-
garis).

Maysin, in seeds of Maize (Zea Mays).

Tuberin, in tubers of Potato (Solanum tuberosum).

vin] THE PROTEINS AND PROTEASES 127

Crystalline globulins have also been isolated from the following
seeds but have as yet no distinctive names: Flax (Linum usitatissimum),
Squash (Cucurbita maxima), Castor-oil Bean (Ridnus communis), Coconut
(Cocos nucifera), Cotton-seed (Gossypium herbaceum), Sunflower (Heli-
anthus annuus), Radish (Raphanus sativus), Peanut (Arachis hypogaea),
Rape (Brassica campestris) and Mustard (Brassica alba).

It will be seen that the majority of reserve proteins of seeds are
globulins. It is probable that native and artificial crystalline proteins
are identical in many cases.

Prolamins. These proteins are characterized by the fact that they
are insoluble in water and dilute saline solutions, but are soluble in
70-90 °/o alcohol. Such proteins are peculiar to plants, and are formed
to a considerable extent in the seeds of cereals. The principal ones
which have been isolated are :

Gliadin found in the seeds of Wheat (Triticum vulgare).

„ „ „ Rye (Secale cereale).

Hordein „ „ Barley (Hordeum vulgare).

Zein • „ „ Maize (Zea Mays).

The properties of the gliadins are demonstrated in Expts. 128, 129,
130 and 131.

Glutelins. The proteins of this group are insoluble in water, dilute
saline solutions and in alcohol, but they are soluble in dilute alkalies.
Glutenin of wheat is the only well-characterized member of this class
which has so far been isolated, though other cereals most probably
contain similar proteins. A protein of this nature has also been obtained
from seeds of Rice (Oryza sativa). The properties of the glutelins are
demonstrated in Expts. 128, 129 and 131.

Nucleoproteins. Though these proteins probably form constituents
of all cells, the only members of the class investigated are those of the
wheat embryo. This has been possible since nuclei form a large pro-
portion of the tissue of the embryo. They may be regarded as protein
salts of nucleic acid, i.e. protein nucleates. On hydrolysis with acids or
enzymes they split up into various proteins and nucleic acid. The
nucleoproteins are also connected with the purins (see p. 164).

Metaproteins. These are hydrolytic products of albumins and glo-
bulins formed by the action of water or dilute acid or alkali. They
are insoluble in water, strong mineral acids and all solutions of neutral
salts, but are soluble in dilute acids and alkalies in the absence of any
large amount of neutral salt.

128 THE PROTEINS AND PROTEASES [CH.

Expt. 125. Reactions of metaprotein. Dissolve about 1 gm. of edestin (see Expt. 133)
in 50 c.c. of a 2 % hydrochloric acid and keep on a boiling water-bath for 2 hrs.
Neutralize with dilute sodium carbonate solution. A copious precipitate of meta-
protein separates out which is insoluble in water. Filter off the precipitate and
wash. Make with it the following tests :

(a) Dissolve up some of the precipitate again in 0-4 °/0 hydrochloric acid. To
portions of the solution add : (i) Dilute sodium carbonate : the .metaprotein is pre-
cipitated again and redissolves in excess, (ii) Concentrated hydrochloric acid : the
metaprotein is precipitated, (iii) Boil some of the acid solution. No coagulum is
formed : the metaprotein is not precipitated by boiling when in solution, and can
still be precipitated by neutralizing with sodium carbonate.

(6) Suspend some of the precipitate in water and boil. Cool and add 0'4 °/0
hydrochloric acid : the precipitate is now insoluble, since the metaprotein is coagu-
lated when boiled in suspension.

(c) To some of the precipitate suspended in water, add gradually saturated
ammonium sulphate solution : the metaprotein is insoluble in all concentrations of
the salt.

Proteoses (albumoses) and peptones. These substances are formed
as products of hydrolysis by enzymes. When present in extracts from
seeds, however, it is sometimes uncertain whether they formed original
constituents of the seeds or resulted from hydrolysis.

As a result of the enzyme hydrolysis of proteins a mixture of various
proteoses is usually produced (Chittenden and Mendel, 4) which can be
separated by various methods, such as different solubilities in ammonium
sulphate, alcohol, etc. The albumoses are soluble in water, salt solutions,
dilute acids and alkalies. They are all precipitated by complete satura-
tion with ammonium sulphate, and some by half-saturation with the
same salt. On the whole, they give the general colour reactions of the
proteins, and are precipitated by the protein precipitants, though some
groups of proteoses show certain exceptions. TJieir solutions are not
coagulated on boiling.

The peptones are the only proteins not precipitated by complete
saturation with ammonium sulphate. They give the protein colour
reactions and are precipitated by tannic acid and lead acetate.

Expt. 126. Separation and reactions of proteoses. Take 2 gms. of the globulin
edestin (prepared as in Expt. 133) and put in a flask with 100 c.c. of 0'2% hydro-
chloric acid and warm until as much as possible of the edestin goes into solution.
Then cool and add 0'5 gm. of commercial pepsin : add also a little toluol, shake
and plug with cotton-wool. Leave in an incubator at 38° C. for 4 days. (A control
experiment should also be made with 100 c.c. of 0'2 °/0 hydrochloric acid and
0'5 gm. of pepsin. Since pepsin itself gives a biuret reaction, a control is necessary
for comparison in the next experiment.) The preliminary changes in edestin hydro-
lysis are rapid, for it will be found that if the edestin solution is tested with sodium

vm] THE PROTEINS AND PROTEASES 129

chloride solution even after 24 hours in the incubator, no precipitate will be given
with sodium chloride, in contrast with the copious precipitate given on the addition
of salt solution to the unaltered acid solution of edestin (see also Expt. 124). After
four days, the incubated mixture is neutralized with dilute sodium carbonate
solution, filtered and saturated while boiling with solid ammonium sulphate.
A precipitate of proteoses is formed, which can be gradually collected together as
a sticky mass and removed with a glass rod. Dissolve the precipitate in some hot
water, filter and make the following tests :

(a) Xanthoproteic reaction. Add a few drops of nitric acid. It is characteristic
of most proteoses that a precipitate is formed which disappears on heating and
reappears on cooling. In the case of the proteoses from edestin, only a slight pre-
cipitate may be given, but it is increased by adding a little sodium chloride solution.
The colour is intensified in the usual way by addition of ammonia.

(b) Millon's reaction. A positive result is given.

(c) Glyoxylic reaction. A positive result is given.

(d) Biuret reaction. A pink or pinkish-violet colour is given.

(e) Sulphur reaction. A positive result is given.

(/) Add a little tannic acid solution. A precipitate is formed.

(<7) Add a drop of copper sulphate solution. A precipitate is formed.

(Ji) Add a drop of strong acetic acid and then a couple of drops of potassium
ferrocyanide. A precipitate is formed which disappears on heating and reappears
on cooling.

(i) Boil some of the solution. No coagulum is formed.

Expt. 127. Detection of peptone. The saturated solution, from which the proteoses
have been precipitated, is then filtered and to a measured quantity (about 5 c.c.)
twice the volume of 40 % sodium hydroxide is added and a drop of 1 °/0 copper
sulphate solution. A pink colour appeal's, due to the presence of peptone. A test
should be made with the control solution containing hydrochloric acid and pepsin
only. An adequate amount should be saturated with ammonium sulphate, filtered
and 5 c.c. tested for peptone. The reaction is less marked than in the actual hydro-
lytic product. Concentrate the remainder of the peptone solution on a water- bath
and pour off from the excess of ammonium sulphate crystals. Filter and make
the following tests : (i) Xanthoproteic, (ii) Millon's, (iii) Glyoxylic, (iv) Tannic acid.
A positive result is obtained in each case.

THE SEED PROTEINS OF CERTAIN PLANTS.

The proteins present in the seeds of certain genera and species, upon
which special investigations have been made, may now be considered.

It should be borne in mind that there are always several proteins
present in the seed. Some are reserve proteins of the cells of the
endosperm or of the storage tissue of the cotyledons : others are proteins
of the protoplasm and nuclei of the tissues of the embryo and of the
endosperm.

o. 9

130 THE PROTEINS AND PROTEASES [CH.

PROTEINS OF CEREALS (GRAMINACEAE).

As far as investigations have gone it may be said that the starchy
seeds of cereals are poor in albumins and globulins. The chief reserve
proteins belong to the peculiar group of prolamins, and a considerable
portion also consists of glutelins.

The grain of Wheat (Triticum vulgare) contains some proteose and
a small percentage of an albumin, leucosin. A globulin occurs only
in very small amount. The bulk of the protein consists of gliadin (a
prolamin) and of glutenin (a glutelin). Nucleoproteins are present in
the embryo, but there is no gliadin or glutenin (Osborne and Voorhees, 14).

Expt. 128. Extraction of the proteins of the Wheat grain, (a) Extraction of
albumin (leucosin) and proteose. Take 100 gms. of white flour (the same quantity of
wheat grains which have been ground in a coffee-mill may be used, but the extraction
in this case is slower), put the ground mass in a large flask or beaker and add
250 r.c. of distilled water. Let the mixture stand for 1-4 hrs., shaking occasionally.
Filter off' some of the liquid, first through muslin and then on a filter-pump. Reserve
the residue on the filter and test the filtrate for proteins [Expt. 121, (a)-(e?)].

Boil a second portion of the filtrate (after adding a drop or two of acetic acid).
A precipitate of the albumin, leucosin, is formed. Filter off this precipitate, cool the
filtrate and make the protein tests again. All the above tests are given by the proteose
in solution : in the case of the xanthoproteic, the precipitate disappears on heating
and reappears on cooling (Expt. 126). Also make the following special test for
proteoseis (Expt. 126) : Add a little potassium ferrocyanide solution and acetic acid.
A white precipitate is formed which disappears on heating and reappears on cooling.

(b} Extraction of the globulin. Take the residue of ground wheat and drain on
a filter-pump. Then extract with 250 c.c. of 10 % sodium chloride solution for
12-24 hrs. Filter off, first through muslin, and then through paper on a filter-
pump. Put the extract to dialyze in a collodion dialyzer for 24 hrs. (toluol should
be added to the liquid in the dialyzer). Filter oft' the precipitate, which will be very
slight, and dissolve it in a little 10% sodium chloride. (Though so little globulin is
present, the experiment is instructive for comparison with the large amount of
globulin obtained from many other seeds.) Make the tests for protein [Expt. 121,
(a}-{d}~\ with the solution (Millon's cannot be used on account of the presence of
chlorides). Also try the effect of (i) boiling the sodium chloride solution : coagulation
is not complete, (ii) adding a little acid : a precipitate is formed as in the case of
edestin.

(c) Extraction of gliadin. Take the wheat residue, which has been filtered from
the sodium chloride solution, and add 250 c.c. of 95 °/0 alcohol. Warm on a water-
bath and filter. Evaporate the filtrate, which contains gliadin, on a water bath (or
better distil off' the alcohol in vacuo}. When reduced to about half its bulk, take a
little of the filtrate and filter. Divide this filtrate into two parts in test-tubes. To
one add water: to the other absolute alcohol. A white precipitate of gliadin is
formed in each case, since it is insoluble in both water and strong alcohol, though
soluble in dilute alcohol. The remainder of the gliadin extract is evaporated almost

vin] ' THE PROTEINS AND PROTEASES 131

to dryness, and then poured into a large volume of distilled water. A milky precipi-
tate of gliadin is formed which may be made to settle by adding a little solid sodium
chloride and stirring. Filter off the gliadin and dissolve in 10 °/0 acetic acid. With
the solution make the tests for protein [Expt. 121, (a)-(d)].

(d) Extraction of glutenin. Take half the wheat residue from the alcoholic ex-
traction, pound well in a mortar and extract again with warm alcohol and subsequently
with water. The residue must be free from water- and alcohol -soluble proteins as
they are also soluble in alkalies. Then extract the residue with O'l % caustic potash
solution. Filter off the extract which contains the glutenin. To a portion of the

N
filtrate add ^- sulphuric acid drop by drop. A precipitate of glutenin is formed.

Test the remainder of the filtrate for proteins [Expt. 121, (a)-(d)].

The gliadin of wheat has the peculiar property of combining with
water to form a sticky mass which binds together the particles of
glutenin, the whole forming what is termed gluten. It is this phenomenon
which gives the sticky consistency and elastic properties to dough.

Expt. 129. To demonstrate the fact that gluten formation depends on the presence
of gliadin. Take two small evaporating dishes. Fill one with ordinary flour. Fill
the other with flour that has been extracted with 70 % alcohol for two or three days.
(The alcohol is allowed to stand on the wheat in the cold. It is then poured off, and
more added, and the process repeated. The flour is now dried again, first in air, then
in the steam-oven and finally is ground in a mortar.) A little water is added to
each of the dishes and the flour worked up into a dough. This is then allowed to
stand for half an hour. The dough consists of gluten (gliadin and glutenin) to which
the starch adheres. Next take two beakers, fill with water, and over the top of each
tie a muslin cover. Place the two samples of dough on the muslin on the two beakers,
and rub gently with a glass rod. The starch will be washed away into the beakers. In
the case of the normal flour a sticky mass of gluten will remain. In the other case
there will be no gluten on account of the absence of gliadin. To the suspension of
starch in the beaker add some iodine solution, and it will turn a deep blue-black colour.

In the Barley (Hordeum vulgare) grain, small percentages of an
albumin, apparently identical with leucosin, and of a globulin, barley
edestin, are present, together with some proteose. The main protein is
a prolamin, hordein, very similar to, but not identical with, gliadin.
There is no well-defined glutelin (Osborne, 9).

Expt. 130. Extraction of the proteins of the Barley grain, (a) Extraction of the
albumin and proteose. Grind up 100 gms. of barley grains in a coffeermill, or use
preferably barley flour. Add 250 c.c. of distilled water to the ground meal, and
allow the mixture to stand for 1-4 hrs. Filter off the extract, first through muslin
and then through filter-paper. The extract will contain a small quantity of the
albumin, leucosin, and proteose. With the filtrate make the tests for proteins
[Expt. 121, (a)-(rf)].

Boil a second portion of the filtrate, A white precipitate of the coagulated pro-
tein is formed. Filter off the precipitate, cool the filtrate containing the proteose

9—2

132 THE PROTEINS AND PROTEASES [CH.

and test for proteins. All the tests will be positive : in the case of the xanthoproteic,
the precipitate disappears on heating and reappears on cooling, a characteristic of
proteoses (Expt. 126). Make also the special test for proteoses : Add a little potas-
sium ferrocyanide and acetic acid. A white precipitate is formed which disappears
on heating and reappears on cooling.

(b} Extraction of the globulin. To the barley residue, after extraction with
water, add 250 c.c. of 10 °/0 sodium chloride and allow the mixture to stand for
12-24 hrs. Filter first through muslin and then filter-paper, and put the extract
to dialyze for 24 hrs. Filter off the precipitate of globulin which will have formed,
and take it up into solution again in as small a quantity as possible of 10 % sodium
chloride. Make with the solution the following tests : (i) The usual (except Millon's)
tests for proteins [Expt. 121, (a)-(d)] : these will give positive results, (ii) Boil a little
of the solution : imperfect coagulation will take place, (iii) Add a little acid : a pre-
cipitate is formed, as is usual with plant globulins.

(c) Extraction of the prolamin, hordein. The residue, after the sodium chloride
extraction, is then extracted with 250 c.c. of warm 95 °/0 alcohol. Filter, and con-
centrate the filtrate on a water-bath (or better, distil in vacuo). After concentration,
test a little filtered extract as follows : pour a few drops into (1) absolute alcohol,
(2) distilled water. A white precipitate of hordein is produced in each case, since,
like gliadin, it is insoluble in both strong alcohol and water, but soluble in dilute
alcohol. Then pour the whole extract into a large volume of water. The protein is
precipitated as a fine white suspension, but will settle out more readily if a little
solid sodium chloride is added. Filter off the hordein, and dissolve in 1 % acetic acid.
Make the usual protein tests [Expt. 121, (a)-(rf)] ; there will be a positive result in
each case.

In the Rye (Secede cereale) grain there are small percentages of
proteose, and of leucosin and edestin. The greater part of the protein
is gliadin, said to be identical with that in wheat.

In the Maize (Zea Mays) grain there is apparently no true albumin,
though there is some proteose. There are small quantities of globulin,
but the greater part of the protein is a prolamin, termed zein, and a
glutenin (Osborne, 10).

Expt. 131. Extraction of proteins of the Maize grain, (a) Extraction of proteins
soluble in water. Grind up 100 gms. of maize grains in a coffee-mill, or preferably use
maize meal. Add 250 c.c. of water and allow the mixture to stand 1-4 hrs. Filter
off, first through muslin, and then filter-paper. The filtrate contains proteose and
probably a little globulin which has gone into solution owing to the presence of salts
in the seed. Make the usual tests for protein [Expt. 121, (a)-(rf)].

Boil another portion of the filtrate. Some coagulation of protein will take place.
Filter, cool the filtrate and test for protein [Expt. 121, (a)-(d)]. Positive results will be
given by the proteose present. Make also the special test for proteoses : Add a little
potassium ferrocyanide and acetic acid. A white precipitate is formed, which dis-
appears on heating and reappears on cooling.

(6) Extraction of globulin. The residue, after water extraction, is next treated .
with about 250 c.c. of 10% sodium chloride solution for 12-24 hrs. Filter, first

vin] THE PROTEINS AND PROTEASES 133

through muslin, and then through filter-paper. Dialyze the extract for 24 hrs. Then
filter off the precipitate of globulin which will have separated out, and dissolve
in 10 % sodium chloride. Make with the solution the following tests : (i) The usual
(except Millon's) tests for proteins [Expt. 121, (a)-(d)] : these will give positive results.
(ii) Boil a little of the solution : imperfect coagulation takes place, (iii) Add a little
acid : the protein is precipitated.

(c) Extraction of the prolamin, zein. The residue after salt extraction is then
extracted with 250 c.c. of hot 95 °/0 alcohol. Filter, and concentrate the filtrate,
which contains the zein, on a water-bath (or, better, distil in vacua). Pour a few
drops of the concentrated extract into (1) absolute alcohol, (2) distilled water. As
in the case of gliadin and hordein, a precipitate of zein will be formed. Then pour
the whole extract, after evaporating to a small bulk, into excess of distilled water,
and add a little solid sodium chloride. The precipitate of zein will slowly settle, and
can be filtered off. Zein is not readily soluble in acids and alkalies. Hence Millon's
and the xanthoproteic tests should be made on the solid material. Zein does not
contain the tryptophane nucleus. To demonstrate this, the glyoxylic reaction should
be made by shaking up some solid zein in reduced oxalic acid and adding sulphuric
acid and mixing. No purple colour is formed.

(d) Extraction of glutenin. Take about half of the residue after the alcoholic
extraction, pound in a mortar, and extract again with alcohol. Then extract the
residue with 0*1 °/0 caustic potash solution. Filter off the extract which contains

the glutenin. To a portion of the filtrate add — sulphuric acid drop by drop.

A precipitate of glutenin is formed. Test the remainder of the filtrate for proteins
[Expt. 121, (a)-(d)].

PROTEINS OF LEGUMINOUS SEEDS (LEGUMINOSAE).

In the Leguminosae, which are starchy seeds, the chief reserve
proteins, as contrasted with those of cereals, are globulins. The various
proteins occurring may be enumerated as :

Legumin. A globulin which forms the chief protein in the seeds of
the Broad Bean (Vida Faba), the Pea (Pisum sativum), the Lentil
(Ervum Lens) and the Vetch (Vicia sativa). Legumin itself is soluble
in water, but occurs as salts which are insoluble in water and soluble
in saline solutions. Some portion can be extracted from the seed by
water only.

Vicilin. A globulin occurring in smaller quantities than legumin
and found only in the Pea, Bean and Lentil seeds.

Phaseolin. A globulin forming the bulk of the protein of the
Kidney Bean (Phaseolus vulgaris).

Conglutin. A globulin forming the bulk of the protein in Lupin
(Lupinus luteus) seeds.

134 THE PROTEINS AND PROTEASES [CH.

Legumelin. An albumin found in small quantities in the Pea,
Broad Bean, Vetch and Lentil.

Phaselin. An albumin found in small quantity in the seeds of the
Kidney Bean (Phaseolus vulgaris).

Small quantities of proteoses are found in most of the above seeds.

Expt. 132. Extraction of the proteins of the Pea (Pisum sativum) (Osborne and
Campbell, 11, 12 ; Osborne and Harris, 13). As we have seen (Expt. 121), a certain
amount of protein, including globulin, goes into solution when ground peas are ex-
tracted with water. A more complete method of extraction is as follows. Grind in
a coffee-mill 20-30 gms. of peas, add to the ground mass 50-60 c.c. of 10 % sodium
chloride solution and allow the mixture to stand for 1-2 hrs. Then filter off and
saturate the filtrate with solid ammonium sulphate. The globulins, legumin and
vicilin, are precipitated out. Filter off the precipitate, and then take up in dilute
ammonium sulphate (y^y saturated) and add saturated ammonium sulphate in the
proportion of 150 c.c. to every 100 c.c. of the solution (j^ saturation). The legumin
is precipitated and can be filtered off. Saturate the filtrate with ammonium sulphate :
the vicilin is precipitated and can be filtered off. Dissolve up a little of each preci-
pitate in 10 % sodium chloride, and boil. The vicilin is coagulated, but the legumin is
not. Then dissolve up the remainder of the precipitates in dilute ammonium sulphate*
and test both the solutions for protein by the usual reactions [Expt. 121, (a)-(d}~\.

The albumin, legumelin, which occurs only in small quantities in the seeds, can
be obtained by dialyzing a water extract of the ground peas until all the globulin is
precipitated. On filtering and heating the filtrate, a coagulum of legumelin is formed.

PROTEINS OF FAT-CONTAINING SEEDS.

Of the seeds which contain fat as a reserve material, those investi-
gated have been found, in contrast to the cereals, to contain largely
globulin as reserve protein. In many cases these globulins have been
obtained in crystalline form after extraction from the plant.

The Hemp-seed (Cannabis sativa) contains one of the best-known
crystalline globulins, namely edestin. Pure neutral edestin is insoluble
in water but soluble in salt solutions. In the presence of acid, however,
edestin forms salts which are insoluble in salt solutions. Hence a
solution of edestin in sodium chloride is precipitated by even small
quantities of acids, and, conversely, a solution of edestin in acid is
precipitated by small quantities of salt (Osborne, 8).

Expt. 133. Extraction and crystallization of edestin from Hemp-seed. Take 50 gms.
of hemp-seed and grind in a coffee-mill. Put the ground seed in a large evaporating
dish and add 200 c.c. of 5 % sodium chloride solution. Heat with a small flame and
stir constantly. A thermometer should be kept in the dish, and the liquid must not
rise above 60° C. Filter off, in small quantities at a time, keeping the solution in the
dish warm. On cooling, the edestin separates out from the filtrate more or less in

vni] THE PROTEINS AND PROTEASES 135

crystals. To obtain better crystals, filter off the edestin that has been deposited, and
pour the filtrate into a dialyzer; add a little toluol, and suspend the dialyzer in
running water. As soon as it is cloudy, examine the dialyzed solution for crystals '
under the microscope. Add a little 5% sodium chloride solution to the original
precipitate of edestin in the filter. Make with the filtrate the following tests:
(i) The tests for proteins [Expt. 121, (a)-(d), except Millon's]. (ii) Boil a little of the
solution : it is imperfectly coagulated, (iii) Add a little acid : edestin chloride is
precipitated.

In the Castor- oil seed (Ricinus communis) there is also present a
globulin which can be obtained in a crystalline form by the method of
Expt. 134. In addition, there is present an albumin, ricin, which has
peculiar toxic properties (Osborne, 8).

A well-crystallized globulin can be obtained from the Linseed (Linum
usitatissimum), and a globulin, excelsin, from the Brazil nut (Bertholletia
excelsa) also in crystalline or semi-crystalline form. Similar globulins
can be extracted from a number of other seeds, i.e. Coconut (Cocos
nucifera), Sunflower (Helianthus annuus), Cotton-seed (Gossypium her-
baceum), Mustard-seed (Brassica alba) and many others. The fat is first
removed from the ground seed by ether or benzene ; the residue is then
extracted with dilute sodium chloride and the extract dialyzed.

Expt. 134. Extraction of the globulin from Ricinus. Weigh out about 50 gins, of
Ricinus seeds, take off the testas and pound in a mortar. Extract the oil by the
method given in Expt. 82. After extracting the oil, grind up the residue again in a
mortar, and then treat it with about twice its bulk of 10 % sodium chloride solution
for 6-12 hrs. Filter successively through muslin and filter-paper and dialyze the
filtrate. The globulin will be precipitated in semi-crystalline spheroids. When
the bulk of the globulin has separated out, filter off the precipitate, and dissolve it
in as dilute a sodium chloride solution as possible. Make the following tests with
the solution : (i) The tests for proteins [Expt. 121, (a)-(d\ except Millon's]. (ii) Boil
a little of the solution : the coagulation is not complete, (iii) Add a little hydrochloric
acid : a precipitate is formed.

Expt. 135. Extraction of the globulin from Linseed (Osborne, 7, 8). Weigh out
about 50 gms. of Linseed and grind it in a coffee-mill. Extract the oil as in Expt. 82.
Treat the residue with about twice its bulk of 10 % sodium chloride solution for
6-12 hrs. Then filter through muslin and filter-paper, and dialyze the filtrate.
The globulin separates out in octahedra. Filter off the protein, and take up in
dilute sodium chloride. Test the solution as in the case of Ricinus globulin in the
previous experiment.

Expt. 136. Extraction of the globulin (excelsin) from the Brazil nut (Osborne, 8).
Weigh out about 100 gms. of the nut, free from the testas, and, after pounding in a
mortar, extract the oil by the usual method. Then proceed as in the two previous
experiments. The protein separates out in semi-crystalline spheroids. Filter off the
precipitated excelsin, and dissolve in dilute sodium chloride solution. Make with
it the tests as for the globulins in the last two experiments.

136 THE PROTEINS AND PROTEASES [CH.

THE AMINO-ACIDS.

•

There is every reason to believe, since they always arise in hydrolysis
of proteins, that amino-acids are universally distributed in the plant.
It is, however, difficult to isolate and detect them, except in certain
special cases, as, for instance, in germinating seeds when a large stor^
of protein is being rapidly hydrolyzed and translocated. The following
is a short account of the occurrence of some of the amino-acids in the
free state (see also p. 120).

Valine has been isolated from seedlings of the Vetch (Vicia), Lupin
(Lupinus) and Kidney Bean (Phaseolus). It is present in larger amounts
in etiolated seedlings of Lupin than in the green plants.

Leucine is widely distributed. It has been isolated from seedlings
of Vicia, Vegetable Marrow (Cucurbita), Lupinus, Pea (Pisum) and
Goose foot (Chenopodium). It has also been found in Phaseolus, Water
Ranunculus (Ranunculus aquatilis), buds of Horse Chestnut (Aesculus
Hippocastanum) and in small quantities in Potato tubers and other
plants.

Isoleucine has been extracted from seedlings of Vicia sativa.
Aspartic acid. The amide of this acid, i.e. asparagin,

CONH2 • CH2 ' CHNH2 • COOH

is widely distributed in plants. It is present in shoots of Asparagus
from which it derives its name. It has also been extracted in very
considerable quantities from etiolated seedlings of Vicia, Lupin, and from
various plants such as Potato, Dahlia, Garden Nasturtium (Tropaeolum\
Cucurbita and Sunflower (Helianthus).

Glutaminic acid. The amide, again, of this acid, i.e. glutamine,

CONH2 • CH2 ' CH2 • CHNH2 • COOH

is widely distributed. It has been isolated from seedlings of Cucurbita,
Lupinus, Helianthus, Castor-oil plant (Ricinus), Spruce Fir (Picea ecccelsa)
and a number of Cruciferae.

Arginine has been isolated from seedlings of Lupinus, Cucurbita,
Vicia, and Pisum. It is especially abundant in the seedlings of some
Coniferae, i.e. Picea excelsa, Silver Fir (Abies pectinata) and Scotch Fir
(Pinus sylvestris}. It also occurs in roots and tubers, as for instance in
those of the Turnip (Brassica campestris), Artichoke (Helianthus tubero-
sus), Chicory (Cichorium Intybus), Beet (Beta vulgaris), Potato and
Dahlia, and in the inner leaves of the Cabbage (Brassica oleracea).

vm] THE PROTEINS AND PROTEASES 137

Lysine has been isolated from seedlings of Lupinus, Vicia and
Pisum. Also from the inner leaves of the Cabbage and tubers of the
Potato.

Phenylalanine has been isolated from seedlings of Lupinus luteus,
Vicia sativa and Phaseolus vulgaris.

Tyrosine is very widely distributed. It is present in seedlings of
Vicia sativa, Cucurbita, Lupinus, Tropaeolum and tubers of Potato,
Turnip, Dahlia, Beet and Celery. Also in berries of Elder (Sambucus),
in Clover (Trifolium), Bamboo (Bambusa) shoots and other plants.

Proline has been isolated in very small quantities from etiolated
seedlings of Lupinus albus.

Histidine has been isolated from seedlings of Lupinus and tubers of
Potato.

Trypt6phane has been isolated from seedlings of Lupinus albus and
Vicia sativa.

THE PROTEASES.

We have seen in the previous pages that proteins can be hydrolyzed
artificially with the intermediate production of proteoses and peptones,
and the final production of a number of amino-acids. There is no doubt
that this process of hydrolysis takes place in the living plant, and it is
believed that the converse process, the synthesis of these proteins from
amino-acids. also takes place in the cell.

There is evidence that this hydrolysis of proteins is catalyzed by
certain enzymes which have been termed proteases. On analogy with
other enzymes, we may suppose that these enzymes also catalyze the
synthesis of the proteins.

It seems highly probable that the proteases are of two types :

1. Pepsin-like enzymes, which catalyze the hydrolysis of proteins to
peptones, and, in all probability, the reverse process.

2. Erepsin-like enzymes, which catalyze the hydrolysis of albumoses
and peptones to amino-acids, and, in all probability, the reverse process.

We now turn to the evidence for the existence of proteases. In
autolysis (see p. 19) the hydrolytic activity of many enzymes is un-
controlled, and in the case of the proteins, the amino-acids are formed
as end-products. Amino-acids are rarely present in plants in sufficient
quantity to be detected readily, at any rate in small quantities of
material, but if the tissues are put to autolyze at temperatures of 38— 40° C.,

138 THE PROTEINS AND PROTEASES [CH.

the amino-acids then accumulate and can be detected. Of all the amino-
acids the one which is most readily identified is tryptophane. If the au tolyzed
product is boiled, acidified and filtered to remove the remaining proteins,
and, to the filtrate, bromine is added, drop by drop, the formation of a
pink or purple colour will indicate the presence of free tryptophane, and
hence it may be assumed that protein-hydrolysis has taken place.
Probably the formation of amino-acids in autolysis is a universal property
of plant tissues, for tryptophane has been detected on autolysis of many
different parts of plants. Examples are the germinating seeds of the
Bean (Vicia Faba), Scarlet Runner (Phaseolus multiftorus}, Pea (Pisum
sativum), Lupin (Lupinus hirsutus) and the Maize (Zea Mays) : and in
ungerminated seeds of the above, though less readily. It is also said to
be formed on autolysis of leaves of Spinach (Spinacia), Cabbage (Brassica),
Nasturtium (Tropaeolum majus), Scarlet Geranium (Pelargonium zonale),
Dahlia (Dahlia variabilis) and others : also of fruits of Melon (Cucumis
Melo), Cucumber (Cucumis sativus), Banana (Musa sapientum), Tomato
(Ly coper sicum esculentum) and others: of bulbs of the Tulip (Tulipa),
Hyacinth (Hyacinthus orientalis) and underground roots of Turnip
(Brassica), Carrot (Daucus Carota) and Beet (Beta vulgaris) (Vines,
16-24; Blood, 3; Dean, 5, 6).

Expt. 137. The formation of tryptophane on autolysis of resting seeds. Grind up
in a coffee-mill 15 gms. of Mustard (Brassica alba] seed. Transfer to a flask, and add
100 c.c. of distilled water and about 2c.c. of toluol. Plug the mouth of the flask
with cotton-wool and put in an incubator for 3 days. Then filter off' the liquid, boil
the filtrate and add a few drops of acetic acid. Filter off any precipitate formed,
cool the filtrate and add bromine water slowly and carefully drop by drop, shaking
well after each drop. A pink or purple colour denotes the presence of tryptophane.
Excess of bromine will destroy the colour. Then shake up with a little amyl
alcohol. The purple colour will be extracted by ttye amyl alcohol which will rise to the
top of the water solution. A control experiment should be made using 10 gms. of
seed which has been well boiled with water in an evaporating dish.

It has been assumed that the formation of amino-acids from proteins
on autolysis is the outcome of two processes, the hydrolysis of proteins
to peptones by pepsins, and the hydrolysis of peptones to amino-acids
by erepsins.

The next point to be considered is the possibility of detecting these
two classes of enzymes separately. If either the pulp, or water extract,
of various plant tissues be added to peptone solution and allowed to
incubate at 38° C., tryptophane can be readily detected after a day or
two. This has been found to be true for the tissues of many seeds,
seedlings, roots, stems, leaves and fruits (such as those already mentioned

vm] THE PROTEINS AND PROTEASES 139

above and others); the result indicates the wide distribution of an
erepsin type of enzyme. The detection of this enzyme is facilitated
by the addition of the artificial supply of peptone.

Expt. 138. The detection of erepsins in plants.

(a) In resting seeds. Grind up 10 gms. of seeds in a coffee-mill, and add 100 c.c.
of water, 0'2 gm. of Witte's peptone and a little toluol. Incubate for 2-3 days. The
following seeds may be used : Hemp (Cannabis sativa), Castor-oil (Ricinus corn-munis'),
Pea (Pisum sativum), Scarlet Runner (Phaseolus multiflorus), Broad Bean (Vicia
Faba) and fruit of Wheat (Triticum vulgare). Test for tryptophane. Controls
should be made in these and the following cases.

(6) In germinating seeds. Take 10 germinating peas, pound in a mortar, add
100 c.c. of distilled water, 0*2 gm. of Witte's peptone, and a little toluol. Incubate
for 3 days. Test for tryptophane.

(c) In leaves. Pound up a small cabbage leaf, add 100 c.c. of water, 0*2 gm. of
Witte's peptone and a little toluol. Incubate for 3 days. Test for tryptophane.

(cF) In roots. Pound up about 20 gms. of fresh carrot root. Add about 100 c.c. of
water, 0'2 gm. of Witte's peptone and a little toluol. Incubate for 3 days. Test for
tryptophane.

The pepsin type of enzyme is less readily detected. It has long
been known that the pitchers of the Pitcher-plant (Nepenthes) secrete
an enzyme which digests fibrin. A few other cases of protein-digesting
enzymes are well known, such as the so-called " bromelin " from the fruit
of the Pine-apple (Ananas sativus), "cradein " from the latex and fruit of
the Fig (Ficus) and " papain " from the fruit and leaves of the Papaw
Tree (Carica Papaya). Such enzymes were formerly termed "vegetable
trypsins" as they were thought to be of the type of animal trypsin which,
alone, hydrolyzes proteins to amino-acids. On analogy with the results of
research with other enzymes, it seems likely that " papain," " cradein "
and u bromelin " are all mixtures of pepsin and erepsin. In addition to
these better known cases, it has also been stated that fibrin is digested
by extracts or pounded pulp of the fruits of the Cucumber and the
Melon, the " germ " (embryo) of Wheat, the bulbs of Tulip and Hyacinth,
the seedlings of the Bean, Pea, Scarlet Runner, Lupin and Maize, and
the unger minated seeds of the Pea, Lupin and Maize. These have also
been shown to contain erepsin.

A separation of pepsin from erepsin has been achieved in the case
of the seeds of the Hemp (Cannabis sativa) by means of the different
solubilities of the two enzymes in water and salt solutions.

Expt. 139. The extraction and the separation of the two enzymes, erepsin and
pepsin, from Hemp-seed (Cannabis sativa) (Vines, 22). Weigh out 50 gms. of hemp-seed,
grind it in a coffee-mill and extract with 250 c.c. of 10% sodium chloride solution.

140 THE PROTEINS AND PROTEASES [CH.

Allow the mixture to stand all night and then filter. Both operations should be
carried out at as low a temperature as possible. Measure the filtrate, and add acetic
acid to the extent of O2 %. A dense precipitate is formed. Filter again, keeping as
cool as possible.

The acid filtrate contains the erepsin, but not the pepsin. Measure out 40 c.c.
into each of three small flasks, and add the following : (i) O2 gm. of Witte's
peptone, (ii) the same, only boil the whole solution, (iii) 0'2gm. of carmine fibrin1.
Add a little toluol to all three flasks, plug with cotton-wool, and incubate for three
to four days. Test for tryptophane in flasks (i) and (ii) ; the first gives a marked
reaction, the second little or no reaction. The fibrin in (iii) will remain unaltered.

The precipitate produced by the acetic acid is then washed on the filter twice
with 100 c.c. of 10 % sodium chloride solution, containing 0'2 °/0 acetic acid, to
remove traces of erepsin. The precipitate is then treated with about 70 c.c. of
water, allowed to stand for a time, and then filtered. The filtrate is divided into
three equal portions. Add the following respectively : (i) 0*1 gm. of carmine fibrin,
(ii) the same, but the solution is boiled, (iii) 0*2 gm. of Witte's peptone. Add a
little toluol to all three flasks, plug with cotton-wool and incubate for 3-4 days.
The fibrin will be seen to digest slowly in flask (i) : (ii) will show no digestion, and
(iii) will give no tryptophane reaction.

REFERENCES

BOOKS

1. Abderhalden, B. Biocheuiisches Handlexikon, iv. Berlin, 1911.

2. Osborne, T. B. The Vegetable Proteins. London, 1909.

PAPERS

3. Blood, A. P. The Erepsin of the Cabbage (Brassica oleracea). J. Biolog.
Chem., 1910-1911, Vol. 8, pp. 215-225.

4. Chittenden, R. H., and Mendel, L. B. On the Proteolysis of Crystallized
Globulin. J. Physiol., 1894, Vol. 17, pp. 48-80.

. 5. Dean, A. L. On Proteolytic Enzymes. I. Sot. Gaz., 1905, Vol. 39,
pp. 321-339.

6. Dean, A. L. On Proteolytic Enzymes. II. Bot. Gaz., 1905, Vol. 40,
pp. 121-134.

7. Osborne, T. B. Proteids of the Flax-seed. Amer. Chem. J., 1892, Vol. 14,
pp. 629-661.

8. Osborne, T. B. Crystallised Vegetable Proteids. Amer. Chem. J., 1892,
Vol. 14, pp. 662-689.

9. Osborne, T. B. The Proteids of Barley. J. Amer. Chem. Soc., 1895, Vol. 17,
pp. 539-567.

1 Freshly washed and finely chopped fibrin is placed in carmine solution (1 gm. carmine,
1 c.c. of ammonia, 400 c.c. of water) for 24 hrs. Then strain off and wash in running
water till washings are colourless.

vni] THE PROTEINS AND PROTEASES 141

10. Osborne, T. B. The Amount and Properties of the Proteids of the Maize
Kernel. J. Amer. Chem. Soc., 1897, Vol. 19, pp. 525-532.

11. Osborne, T. B., and Campbell, G. P. Proteids of the Pea. J. Amer.
Chem. Soc., 1898, Vol. 20, pp. 348-362.

12. Osborne, T. B., and Campbell, G. F. The Proteids of the Pea, Lentil,
Horse Bean and Vetch. J. Amer. Chem. Soc., 1898, Vol. 20, pp. 410-419.

13. Osborne, T. B., and Harris, I. P. The Proteins of the Pea (Pisum
sativum}. J. Biol. Chem., 1907, Vol. 3, pp. 213-217.

14. Osborne, T. B., and Voorhees, C. G. The Proteids of the Wheat-
Kernel. Amer. Chem. J., 1893, VoL 15, pp. 392-471.

15. Reeves, G. A new Method for the Preparation of the Plant Globulins.
Biochem. J., 1915, Vol. 9, pp. 508-510.

16. Vines, S. H. Tryptophane in Proteolysis. Ann. Sot., 1902, Vol. 16,
pp. 1-22.

17. Vines, S. H. Proteolytic Enzymes in Plants. I. Ann. Sot., 1903, Vol. 17,
pp. 237-264. II. Ibid. pp. 597-616.

18. Vines, S. H. The Proteases of Plants. I. Ann. Sot., 1904, Vol. 18,
pp. 289-317.

19. Vines, S. H. The -Proteases of Plants. II. Ann. Sot., 1905, Vol. 19,
pp. 149-162.

20. Vines, S. H. The Proteases of Plants. III. Ann. Sot., 1905, Vol. 19,
pp. 171-187.

21. Vines, S. H. The Proteases of Plants. IV. Ann. Sot., 1906, Vol. 20,
pp. 113-122.

22. Vines, S. H. The Proteases of Plants. V. Ann. Sot., 1908, Vol. 22,
pp. 103-113.

23. Vines, S. H. The Proteases of Plants. VI. Ann. Sot., 1909, Vol. 23,
pp. 1-18.

24. Vines, S. H. The Proteases of Plants. VII. Ann. Sot., 1910, Vol. 24,
pp. 213-222.

CHAPTER IX

-i

GLUCOSIDES AND GLUCOSIDE-sVLITTING ENZYMES

ATTENTION has been drawn to the fact (Chapters v and vn) that in the
plant, compounds containing;hydroxyl groups often have one or more of
these groups replaced by the C^HUO5 — residue of glucose. Such com-
pounds are termed glucosides. The substances in which this substitution
most frequently occurs are of the aromatic class, and the glucosides may
be regarded, on the whole, as ester-like compounds of carbohydrates
with aromatic substances. The non-sugar portion of the glucoside may
vary widely in nature, and may be, for instance, an alcohol, aldehyde,
acid, phenol, flavone, etc. The sugar constituent is most frequently
glucose, but pentosides, galactosides, mannosides and fructosides are also
known. Sometimes more than one inonosaccharide takes part in the
composition of the glucoside. (These various relationships are shown in
the accompanying table.) The inclusion of all glucosides in a class is
in a sense artificial: the character held in common (with very few
exceptions) is that, on boiling with dilute acids, or, by the action of
enzymes, hydrolysis takes place, and the glucoside is split up into
glucose (or other sugar) and another organic constituent. A number of
compounds occurring as glucosides have already been dealt with, for
example, the tannins and flavone, flavonol and anthocyan pigments, but,
in these cases, the significance of the compounds lies rather in the
nature of their non-sugar constituents than in the fact of their being
glucosides. '

There are, however, a number of glucosides which have been grouped
together and are more readily classified in this way than in any other.
Some of them, doubtless, have come into prominence as glucosides on
account of their association with well-known and specific enzymes, as,
for instance, the glucoside amygdalin associated with the enzyme emul-
sin, and the glucoside sinigrin with the enzyme myrosin.

The hydrolyzing enzymes are by no means always specific, for in
vitro one particular enzyme may be able to hydrolyze several glucosides.
Many glucoside-splitting enzymes have been described, though there is
no reason to suppose that each glucoside is only acted upon by an
enzyme specific to that glucoside. It is likely moreover that some of
the different enzymes described will probably prove to be identical.

In some cases where more than one monosaccharide is attached to

CH. IX]

GLUCOSIDE-SPLITTING ENZYMES

143

the glucoside, the different sugar groups are removed separately by
different enzymes (see later, emulsin, p. 145).

The glucosides as a whole (except flavone, flavonol and anthocyan
pigments) are colourless crystalline substances. When extracting them
from the plant, it is usually necessary to destroy the accompanying
enzyme by dropping the material into boiling alcohol or some other
reagent (see autolysis, p. 19).

In Chapter v it has already been mentioned that d-glucose exists in
two stereoisomeric forms, the a. and the /3 form.

It was also pointed out that the glucosides can be classed either as
a- or /3-glucosides, according to whether the a or the ft form of glucose
combines with the non-glucose residue.

RO— C^-H H— C-OR

H— C— OH\^ H— C—OH

HO-C— H

HO— C— H

H—

H— C— OH

I

H—

H— C— OH

CH2OH CH2OH

a-glucoside /8-glucoside

Maltose, for instance, is regarded as an a-glucoside of rf-glucose. It
has been further shown that the enzyme maltase can only hydrolyze
a-glucosides, whereas other enzymes, e.g. the prunase component of
emulsin, only act on /3-glucosides.

The various glucosides considered in detail in this chapter together
Avith some others are grouped under the following headings (Arm-
strong, 3) :

Glucoside Plant in which commonly

found

Coniferin (Coniferae, Beta, Asparagus,

Scorzonera}
Populin (Populus)

Salioin (Salix, Populus}

Syringin (Ligustmm, Syringa, Jasmi-

num)

Amygdalin (Prunus, Pyrus)

Dhurrin (Sorghum}

Lmamarin (Linum, Phaseolus]

Products of hydrolysis

Alcohols
Glucose + coniferyl alcohol

Glucose + saligenin -f benzoic acid
Glucose + saligenin
Glucose + syringenin

Aldehydes
Glucose + benzaldehyde -f prussic

acid
Glucose + parahydroxybenzaldehyde

+ prussic acid
Glucose + acetone 4- prussic acid

144

Glucoside

Prulaurasin
Prunasin
Sambunigrin
Vicianin

Gaultherin
Strophanthin

Arbutin
Hesperidin
Naringin
Phloridzin

Aesculin
Fraxin

Glucotropaeolin

Sinalbin

Sinigrin

Apiin

Isoquercitrin

Lotusin

Myricitrin

Quercitrin

Robinin

E-utin

Cyanin
Delphinin

Malvin
Oenin
Peonin
Pelargonin

Aucubin
Digital in
Indican

GLUCOSIDES AND

[CH.

Plant in which commonly
found

(Prunus)

(Cerasus, Prunus)
(Sambucus)
( Vicia)

(Gaultheria, Spiraea}
(Strophanthus)

(Ericaceae)
(Citrus)
(Citrus}
(Rosaceae)

(Aesculus)
(Fraxinus)

( Tropaeolum, Lepidium}
(Brassica alba)
(Brassica nigra)

(Carum)

(Gossypium)

(Lotus)

(Myricd)

(Quercus, Fraxinus^ Thed}

(Kobinia)

(Ruta, Capparis, Polygonum}

(Centaurea, Rosa)
(Delphi?

(Malva)

( Vitis)

(Paeonid)

(Pelargonium, Centaurea)

(Aucuba, Plantago)

(Digitalis)

(Indigo/era)

Products of hydrolysis

Aldehydes (cont.)
Glucose -f benzaldehyde -I- prussic

acid
Glucose + benzaldehyde + prussic

acid
Glucose -f benzaldehyde + prussic

acid
Vicianose + benzaldehyde + prussic

acid

Acids

Glucose -f- methyl salicylate
Mannose + rhamnose + strophanthi-
din

Phenols

Glucose + hydroquinone
Glucose + rhamnose + hesperetin
Glucose -f rhamnose + narigenin
Glucose -f phloretin

Coumarin derivatives
Glucose + aesculetin
Glucose + fraxetin

Mustard-oils
Glucose + benzyl isothiocyanate +

potassium hydrogen sulphate
Glucose + sinapin acid sulphate -f-

acrinylisothiocyanate
Glucose + allyl isothiocyanate +

potassium hydrogen sulphate

Flavbne andjlavonol pigments
Apiose + apigenin
Glucose + quercetin
Glucose + prussic acid + lotofl avin
Rhamnose + myricetin
Rhamnose + quercetin
Rhamnose -f galactose + kaempferol
Glucose H- rhamnose + quercetin

Anthocyan pigments
Glucose + cyanidin
Glucose + oxy benzoic acid + delph i -

nidin

Glucose -|- malvidin
Glucose -foenidin
Sugar + peonidin
Glucose + pelargonidin

Various constituents
Glucose + aucubigeriin
Glucose -h digitalose + digitaligenin
Glucose + indoxyl

ix] GLUCOSIDE-SPLITTING ENZYMES 145

CYANOPHOKIC GLUCOSIDES.

The characteristic of these substances is that they yield prussic acid
as one of the products of hydrolysis. They are fairly widely distributed :
the following list (Greshoff, 15) includes most of the natural orders in
which such glucosides occur: Araceae, Asclepiadaceae, Berberidaceae,
Bignoniaceae, Caprifoliaceae, Celastraceae, Compositae, Convolvulaceae,
Cruciferae, Euphorbiaceae, Graminaceae, Leguminosae, Linaceae,
Myrtaceae, Oleaceae, Passifloraceae, Ranunculaceae, Rhamnaceae,
Rosaceae, Rubiaceae, Rutaceae, Saxifragaceae, Tiliaceae and Urticaceae.

Amygdalin. This is one of the most important of the cyanophoric
glucosides. It occurs in the seeds of the bitter Almond (Prunus
Amygdalas) but it appears to be almost entirely absent from the sweet
or cultivated Almond. It also occurs in the seeds of the other species of
Prunus — the Plum (P. domestica), the Peach (P. Persica), etc. — of the
Apple (Pyrus Mains) and the Mountain Ash (P. Aucuparia). It occurs
sometimes in leaves, flowers and bark.

By the action of an enzyme, originally termed emulsin, which occurs
in both the bitter and the sweet varieties of Almond, the glucoside is
broken up as follows in two stages:

C2oH27NOn + H20 = C6H1206 + CUH17NO6

mandelonitrile glucoside (prunasin)

C14H17N06 + H20 = C6H1206 + HCN + C6H5CHO

benzaldehyde

It should be noted that the sweet Almond contains emulsin although
it is almost entirely free from amygdalin.

Recently (Armstrong, Armstrong and Horton, 8) emulsin has been
shown to consist of two enzymes, amygdalase and prunase : amygdalase
hydrolyzes amygdalin with formation of mandelonitrile glucoside and
glucose, whereas prunase hydrolyzes mandelonitrile glucoside (prunasin)
with formation of benzaldehyde, prussic acid and glucose. On the basis
of these reactions amygdalin is represented as:

C6H5

CH2OH CHOH CH CHOH CHOH CH • O ' CH2CHOH CH CHOH CHOH CH • O • CH

CN

Prunasin occurs naturally in the Bird Cherry (Cerasus Padiis), and
it is found that prunase may exist in a plant, e.g. Cherry Laurel
(P. Laurocerasus), which does not contain amygdalase.

o. 10

146 GLUCOSIDES AND [CH.

Prulaurasin (laurocerasin) is a glucoside occurring in the leaves of
the Cherry Laurel (Primus Laurowasus). It has been represented as
racemic mandelonitrile glucoside, prunasin being the dextro form.

Sambunigrin is a glucoside occurring in the leaves of the Elder
(Sambucas nigra). It has been represented as laevo mandelonitrile
glucoside.

When tissues containing cyanophoric glucosides and their corre-
sponding enzymes are submitted to autolysis, injury, or the action of
chloroform, hydrolysis takes place (see autolysis, p. 19). A rapid
method (Mirande, 17; Armstrong, 5) for detecting the prussic acid is to
insert paper dipped in a solution of sodium picrate into a tube con-
taining the plant material together with a few drops of chloroform. In
the presence of prussic acid the paper becomes first orange and finally
brick-red owing to the formation of picramic acid.

In addition to those previously mentioned there are other British
plants, the leaves of which give off prussic acid on autolysis (presumably
from cyanophoric glucosides), as for example the Columbine (Aquilegia
vulgaris), Arum (Aram maculatum), Hawthorn (Crataegus Oxyacantha),
Keed Poa (Glyceria aquatica), Bird's-foot Trefoil (Lotus corniculatus),
Alder Buckthorn (Rhamnm Frangula), Black and Red Currant and
Gooseberry (Ribes nigrum, R. rubrum, R. Grossularia), Meadow Rue
(Thalictrum aquilegifolium) and the Common and Hairy Vetches ( Vicia
sativa and F. hirsuta).

It has been shown (Armstrong, 7) that of the species L. corniculatus
there is a variety (L. uliginosus) (taller and growing in moister
situations) which does not produce cyanophoric substances and hence
does not give off prussic acid on autolysis.

Expt. 140. Method of detection of cyanophoric glucosides in the plant. Take three
flasks: in one put a whole leaf of the Cherry Laurel (Prunus Laurocerasus] : in the
second a leaf which has been torn in pieces and then either pricked with a needle or
pounded in a mortar : in the third a leaf with a few drops of chloroform. Cork all
three flasks, inserting with the corks a strip of sodium picrate paper. (The paper is
prepared in the following way : strips of filter-paper are dipped in a 1 °/0 solution of
picric acid, are then suspended on a glass rod and allowed to dry in air. Before
using, the paper is moistened with 10% sodium carbonate solution and is suspended
in the moist condition just above the material to be examined. In the presence of
prussic acid, the paper first becomes orange-yellow, then orange and finally brick-red.)
In a short time the paper in the flask containing the leaf and chloroform will turn red :
in the flask with the injured leaf, the reddening will take place rather more slowly,
whereas in the case of the entire leaf, the paper will remain yellow.

The above experiment may also be carried out, usually with success, on leaves of

ix] GLUCOSIDE-SPLITTING ENZYMES 147

the Columbine (Aquilegia vulgaris\ the Arum (Arum ma-culatum) and plants of the
Bird's-foot Trefoil (Lotus comiculatus) : also with bitter almonds and apple pips, and
young shoots of Flax (Linum perenne). In the case of the seeds, these may be used
crushed, both with and without chloroform, the uninjured seed being used as a
control.

Expt. 141. Preparation of amygdalin. Weigh out 100 gms. of bitter almonds.
Remove the testas by immersing them for a short time in boiling water. Then
pound up the almonds well in a mortar and transfer to a flask. Add about
200-300 c.c. of ether and allow the mixture to stand for 2-12 hours. Filter off the
ether and extract again with fresh ether. The greater part of the fat will be removed
in this way. Then dry the residue from ether and, as rapidly as possible, extract
twice or three times with boiling 90-98 % alcohol which removes the amygdalin.
The residue, after ether extraction, contains both amygdalin and emulsin, and, if
allowed to stand, the emulsin will hydrolyze the amygdaliu : hence the necessity for
rapid extraction with alcohol. Evaporate the filtered alcoholic extract on a water-
bath or, better, distil in vacua to a small bulk. Then add an equal volume of ether
and allow the mixture to stand for a time. The amygdalin separates out on standing.
Filter off the precipitate, dissolve in a little hot water and allow to crystallize in a
desiccator.

Expt. 142. Preparation of emulsin (Bourquelot, 10). Weigh out 25 gms. of
sweet almonds. (Bitter almonds can also be used. The sweet variety is preferable ;
since from them the emulsin can be more readily prepared free from amygdalin.)
Plunge them for a moment into boiling water and remove the testas. Pound
thoroughly in a mortar, and extract the bulk of the oil with ether as in the last
experiment. Then grind up the residue with 50 c.c. of a mixture of equal parts of
distilled water and water saturated with chloroform and allow the whole to stand
for 24 hours. Filter by means of a filter-pump, and to the filtrate add glacial
acetic acid (1 drop to 15 esc. of the filtrate) whereby the protein is precipitated.
Again filter, and to the filtrate add 3-4 times its volume of 96-98 % alcohol. The
emulsin is deposited as a white precipitate. Filter off the precipitate and dissolve it
in about 100 c.c. of cold distilled water.

Expt. 143. (a) To demonstrate the hydrolysis of amygdalin by emulsin. Into
each of two flasks put 50 c.c. of a 1-3 % solution of amygdalin. To one flask add
25 c.c. of the emulsin solution prepared in the last experiment. To the other flask
add 25 c.c. of enzyme solution after it has been well boiled, and again boil the
mixture after adding the enzyme. Fit each flask with a cork and sodium picrate
paper. The paper in the flask containing the unboiled enzyme will rapidly turn red,
the control remaining yellow. Unless both the enzyme and the amygdalin solution
are well boiled in the case of the control, the paper may show reddening in time on
account of traces of prussic acid present in both solutions.

(b) Simplified method for extraction of amygdalin and emulsin, and demonstra-
tion of hydrolysis of amygdalin by emulsin. Take 1 2 bitter almonds. Remove the
testas by immersing them for a short time in boiling water. Then pound up the
almonds well in a mortar and transfer to a flask. Add about 50 c.c. of alcohol and
heat to boiling on a water-bath. Filter off the extract, and evaporate it to dryness
on a water-bath. The residue will contain amygdalin.

10—2

148 GLUCOSIDES AND [CH.

Take six sweet almonds and remove the testas as before. Pound in a mortar and
transfer to a flask. Add a little ether and allow to stand for a short time. Pour off
the ether, and add a little more which should again be poured off. This removes some
of the fat and makes extraction of the emulsin easier. Then extract the residue
with about 40c.c. of distilled water and filter. The filtrate contains the enzyme
emulsin. •.

Take 10 c.c. of the emulsin solution, and divide it into two portions in two test-
tubes. Boil one well (see Expt. 143 a), and to both add equal quantities of a water
extract of the amygdalin prepared above. Cork the tubes and insert picric paper
with the cork in each case.

It has been found, as previously mentioned, that emulsin can
hydrolyze other glucosides, as for instance, salicin (see pp. 48, 152). On
hydrolysis, salicin splits up into salicylic alcohol (saligenin) and glucose.
Salicin, itself, gives no colour with ferric chloride but saligenin gives a
violet colour, and by means of this reaction the course of the hydrolysis
can be followed.

Expt. 144. To demonstrate the hydrolysis of salicin by emulsin. To 10 c.c. of a
1 % solution of salicin in a test-tube add 10 c.c. of the emulsin solution prepared in
Expt. 142 or 143. As a control boil in a second test-tube another 10 c.c. of the
emulsin solution and add 10 c.c. of salicin solution. After about an hour, add to
both test-tubes a few c.c. of strong ferric chloride solution. A purple colour will be
given in the first test-tube but no colour in the control. The process of hydrolysis
will be accelerated by placing the tubes in an incubator.

A modification can be made as follows. A second pair of test-tubes should
be prepared as before and to both sufficient ferric chloride should be added to give a
faint yellow tinge. The unboiled mixture will gradually acquire a purple colour at
ordinary temperature.

Other cyanophoric glucosides are dhurrin, phaseolunatin (linamarin),
lotusin and vicianin.

Dhurrin occurs in seedlings of the Great Millet (Sorghum vulgare).
On hydrolysis it yields glucose, prussic acid and parahydroxybenzalde-
hyde (C6H4 • OH - CHO). It is hydrolyzed by emulsin.

Phaseolunatin occurs in seeds of the wild plants of Phaseolus
lunatus and in seedlings of Flax (Linuiri). It is associated with an
enzyme which hydrolyzes it into acetone, glucose and prussic acid.

Lotusin occurs in Lotus arabicus. On hydrolysis by an accompanying
enzyme (lotase) it gives glucose, prussic acid and a yellow pigment,
lotoflavin.

Vicianin occurs in the seeds of a Vetch (Vicia angustifolia). It is
hydrolyzed by an accompanying enzyme into prussic acid, benzaldehyde
and a disaccharide, vicianose.

ix] GLUCOSIDE-SPLITTING ENZYMES 149

. MUSTARD-OIL GLUCOSIDES.

These are glucosides containing sulphur and they have been found
chiefly among the Cruciferae. Sinigrin and sinalbin, the glucosides of
mustard, have been most investigated.

Sinigrin. This glucoside occurs in the seed of Black Mustard
(Brassica nigra) and other species of Brassica. Also in the root of the
Horse-radish (Cochlearia Armoracia). Sinigrin is hydrolyzed by the
enzyme, myrosin (Guignard, 16; Spatzier, 18) (which occurs in the
plant together with the glucoside), into allyl isothiocyanate, potassium
hydrogen sulphate and glucose:

C]0H16O9NS2K -f H2O = C3H5NCS + C6H12O6 + KHSO4

Expt. 145. Extraction of sinigrin from Black Mustard. Weigh out 100 gms. of
Black Mustard seed. Grind the seed in a coffee-mill and afterwards pound in a
mortar. Heat 175 c.c. of 85 °/0 alcohol to boiling in a flask on a water-bath and add
the pounded mustard, and after boiling about \ hour, filter and press out the alcohol.
Then put the dried cake of residue into 300 c.c. of water and allow the mixture to
stand for 12 hours. Press out the liquid and after filtering and neutralizing with
barium carbonate, concentrate in vacuo to a syrup. Then extract with 90 °/0 alcohol
and filter. On concentrating and exposing in a crystallizing dish, the sinigrin
separates out in white needles.

Sinalbin occurs in the seeds of White Mustard (Sinapis alba). By
myrosin it is hydrolyzed to ^-hydroxybenzylisothiocy^anate, acid sinapin
sulphate and glucose:

H2O = C6H12O6 + C7H7ONCS + C,eH24O5NHSO4

Expt. 146. Extraction of sinalbin from White Mustard. Weigh out 100 gms. of
White Mustard seed. Grind and pound well and extract the fat with ether. Then
extract with twice its weight of 85-90 °/0 alcohol several times and well press out the
alcohol. The extract is evaporated to half its bulk and filtered. On cooling the
sinalbin separates out in crystals.

Expt. 147. Preparation of myrosin. Weigh out 50 gms. of White Mustard seed and
grind in a coffee-mill. Add 100 c.c. of water and allow the mixture to stand for
12 hours. Then filter and allow the filtrate to run into 200 c.c. of 95-98 °/0 alcohol.
A white precipitate is formed which contains the myrosin. Filter off the precipitate
and wash on the filter with a little ether.

Expt. 148. Action of myrosin on sinigrin. Put into two test-tubes equal quantities
of a solution of the sinigrin prepared in Expt. 145. Dissolve some of the myrosin
prepared in the last experiment in water and divide the solution into two parts.
Heat one part to boiling and then add the two portions respectively to the two test-
tubes of sinigrin. Plug both test-tubes with cotton-wool. After about ^ hour a
strong pungent smell of mustard oil, allyl isothiocyanate, will be detected in the
unboiled tube.

A more simple method of demonstrating the action of myrosin is as follows.

150 GLUCOSIDES AND [CH.

Pound about 5 grns. of Black Mustard seed in a mortar and then boil with water.
Some mustard oil will be formed before the myrosin is destroyed, so that boiling
should be continued until no pungent odour can be detected. Then filter and cool
the solution and divide into two parts. To one add some myrosin solution. To the
other an equal quantity of boiled enzyme solution. After £ hour the smell of allyl
isothiocyanate should be detected in the unboiled tube.

SAPONINS.

These substances are very widely distributed, being found in the
orders: Araliaceae, Caprifoliaceae, Combretaceae, Compositae, Cucurbi-
taceae, Graminaceae, Guttiferae, Lecythidaceae, Leguminosae, Liliaceae,
Loganiaceae, Magnoliaceae, Myrtaceae, Oleaceae, Piperaceae, Pitto-
sporaceae, Polemoniaceae, Polygalaceae, Primulaceae, Proteaceae,
Ranunculaceae, Rhamnaceae, Rosaceae, Rutaceae, Saxifragaceae,
Thymelaeaceae and the majority of the orders of .the cohort Centro-
spermae. On hydrolysis with dilute mineral acids the saponins yield
various sugars — glucose, galactose, arabinose, rhamnose — together with
other substances termed sapogenins.

The saponins are mostly amorphous substances readily soluble
in water (except in a few cases) giving colloidal solutions. These
solutions froth on shaking, and with oils and fats they produce very
stable emulsions. By virtue of this property they have been used as
substitutes for soap. The Soapwort (Saponaria) owes its name to the
fact that the root contains a saponin.

COUMARIN GLUCOSIDES.

These substances are hydroxy derivatives of coumarin, which itself
may be represented as:

CH-CH— CO

Aesculin is one of the best known of these glucosides. It occurs in
the bark of the Horse Chestnut (Aesculus Hippocastanum). On hydro-
lysis with dilute acids it yields glucose and aesculetin, the latter being
represented as:

CH=CH— CO

HO

ix] GLUCOSIDE-SPLITTING ENZYMES 151

Aesculin is characterized by giving in water solution a blue fluor-
escence which can be detected even in great dilution. The fluorescence
is increased in alkaline, and decreased in acid, solution.

Expt. 149. Demonstration of the presence of aesculin in Aesculus bark. Strip off
the bark from some young twigs of Aesculus and boil in a little water in an evaporating
dish. Filter and pour the nitrate into excels of water in a large veasel. A blue
fluorescent solution will be formed.

GLUCOSIDES OF FLAVONE, FLAVONOL AND ANTHOCYAN PIGMENTS.
These substances have already been considered in Chapter vn.

GLUCOSIDES OF VARIOUS COMPOSITION.

Coniferin. This glucoside occurs in various members of the Coniferae
and also in Asparagus. On hydrolysis with mineral acids or emulsin, it

breaks up as:

CH = CHCH2OH

CLJ f\ iLJrt OUI f">
16n2'2'-'8 "•" n2^-' ~ ^6n12V-'6

Coniferin

\

OH

Coniferyl alcohol

Arbutin. This glucoside is found in the leaves of the Bearberry
(Arctostaphylos Uva-ursi), Pyrola, Vaccinium, and other Ericaceae and
also of the Pear (Pyrus communis}.

On hydrolysis with acids arbutin yields hydroquinone and glucose:
C12H1607 + H20 = C6H602 + C6H1206

the same hydrolysis is brought about by the enzyme emulsin.

It has been suggested that the darkening of leaves of the Pear
(Bourquelot and Fichtenholz, 11, 12, 13) either on autolysis or injury, or
at the fall of the leaf, is due to the hydrolysis of the arbutin by a gluco-
side-splitting enzyme in the leaf, and subsequent oxidation of the
hydroquinone so formed by an oxidase.

Expt. 150. Extraction of arbutin from leaves of the Pear (Pyrus commuuis).
Weigh out 100 gms. of fresh leaves (without petioles). Tear the leaves into small
pieces and drop them as quickly as possible into about 500 c.c. of boiling 96-98 %
alcohol in a flask. Boil for about 20 mins., adding more alcohol if necessary. Then
filter off the alcohol and pound up the leaf residue in a mortar and extract again with

152

GLUCOSIDES AND

[CH.

boiling alcohol. Filter and distil off the alcohol from the extract in vacuo. Extract
the residue with 100-200 c.c. of hot water and filter. Warm the filtrate and precipitate
with lead acetate solution until no more precipitate is formed. This removes flavones,
tannins, etc. but the arbutin is not precipitated. Filter and pass sulphuretted hydrogen
into the filtrate to remove any excess of lead acetate. Filter and concentrate the
filtrate in vacua to a syrup. Then extract twice with small quantities of ethyl acetate.
Concentrate the ethyl acetate on a water-bath and cool. A mass of crystals of arbutin
will separate out. This should be filtered off on a small filter, and recrystallized from
ethyl acetate. Take up a little of the purified glucoside in water and add a drop or
two of ferric chloride solution. A blue coloration will be given.

Salicin. This substance occurs in the bark of various species of
Willow (Salix) and Poplar (Populus): also in the flower-buds of the
Meadow-Sweet (Spiraea Ulmaria). On hydrolysis with acids, or on
treatment with emulsin, salicin is decomposed into saligenin or salicylic
alcohol and glucose:

C13H1807 + H20 - C6H4OH • CH..OH + C6H12O6

Saligenin gives a violet colour with ferric chloride solution and in this
way the progress of the reaction can be demonstrated (see also p. 148).

Indican (see also p. 115). This glucoside occurs in shoots of the
so-called "Indigo Plants," Indigofera Anil, I. erecta, I. tinctoria, I. suma-
trana: also in the Woad (I satis tinctoria), in Polygonum tinctorium and
species of the Orchids, Phajus and Calanthe. When boiled with acid or
hydrolyzed by an enzyme contained in the plant, it gives glucose and
indoxyl :

•0-C6Hn05

+ C6H12Oe

The colourless indoxyl can be oxidized either artificially or by an
oxidase contained in the plant to a blue product, indigotin or indigo.

OH

Indoxyl

Indigo

ix] GLUCOSIDE-SPLITTING ENZYMES 153

REFERENCES
BOOKS

1. Abderhalden, B. Biochemisches Handlexikon, n. Berlin, 1911.

2. Allen's Commercial Organic Analysis. Glucosides (E. F. Armstrong), Vol. 7,
1913, pp. 95-135.

3. Armstrong, B. F. The Simple Carbohydrates and the Glucosides. London,
1919. 3rded.

4. Van Rijn, J. J. L. Die Glykoside. Berlin, 1900.

PAPERS

5. Armstrong, B. F. The Rapid Detection of Emulsin. J. PhysioL, 1910,
Vol. 40, p. xxxii.

6. Armstrong, H. B., Armstrong, B. F., and Horton, B. Studies on
Enzyme Action. XII. The Enzymes of Emulsin. Proc. R. Soc., 1908, B Vol. 80,
pp. 321-331.

7. Armstrong, H. B., Armstrong, B. P., and Horton, B. Herbage Studies.

I. Lotus corniculatus, a Cyanophoric Plant. Proc. R. Soc., 1912, B Vol. 84, pp. 471-484.

II. Variation in Lotus corniculatus and Trifolium repens (Cyanophoric Plants).
Proc. R. Soc., 1913, B Vol. 86, pp. 262-269.

8. Armstrong, H. B., Armstrong, B. F., and Horton, B. Studies on
Enzyme Action. XVI. The Enzymes of Emulsin. Proc. R. Soc., 1912, B Vol. 85.
(i) Prunase, the Correlate of Prunasin. pp. 359-362. (ii) Distribution of 3-Enzymes
in Plants, pp. 363-369. (iii) Linase and other Enzymes in Linaceae. pp. 370-378.

9. Armstrong, H. E., and Horton, E. Studies on Enzyme Action. XIII.
Enzymes of the Emulsin Type. Proc. R. Soc., 1910, Vol. 82, pp. 349-367.

10. Bourquelot, E. Sur 1'emploi des enzymes comme reactifs dans les re-
cherches de laboratoire. J. pharm. chim., 1906, Vol. 24, pp. 165-174 ; 1907, Vol. 25,
pp. 16-26, 378-392.

11. Bourquelot, E., et Fichtenholz, A. Sur la presence d'un glucoside dans
les feuilles depoirieret sur son extraction. J. pharm. cl.im., 1910, Vol. 2, pp. 97-104.

12. Bourquelot, E., et Fichtenholz, A. Nouvelles recherches sur le gluco-
side des feuilles de poirier ; son r61e dans la production des teintes automnales de ces
organes. J. pharm. chim., 1911, Vol. 3, pp. 5-13.

13. Bourquelot, E., et Fichtenholz, A. Sur le glucoside des feuilles de
poirier. C. R. Acad. sci., 1911, Vol. 153, pp. 468-471.

14. Dunstan, W., and Henry, T. A. The Chemical Aspects of Cyanogenesis
in Plants. Rep. Brit. Ass., 1906, pp. 145-157.

15. Greshoff, M. The Distribution of Prussic Acid in the Vegetable Kingdom.
Rep. Brit. Ass., 1906, pp. 138-144.

16. Guignard, L. Sur quelques proprietes chimiques de la myrosine. Bui.
soc. hot., 1894, Vol. 41, pp. 418-428.

17. Mirande, M. Influence exercee par certaines vapeurs sur la cyanogenese
vegetale. Precede rapide pour la recherche des plantes a acide cyanhydrique.
C. R. Acad. sci., 1909, Vol. 149, pp. 140-142.

18. Spatzier, W. Ueber das Auftreten und die physiologische Bedeutung des
Myrosins in der Pflanze. Jahrb. wiss. Bot., 1893, Vol. 25, pp. 39-77.

19. Winterstein, B., und Blau, H. Beitrage zur Kenntnis der Saponine.
Zs. physiol. Chem., 1911, Vol. 75, pp. 410-442.

CHAPTEK X

THE PLANT BASES

THERE are present in plants a number of substances which form a
group, and which may be termed nitrogen bases, or natural bases.
These substances are of various constitution but they have the property
in common of forming salts with acids by virtue of the presence of
primary, secondary, or tertiary amine groupings. Such groupings confer
a basic property upon a compound and, as a result, salts are formed
with acids on analogy with the formation of ammonium salts:
NH3 + HCI = NH4CI (NH3 • HCl)

CH3NH2+HCI = CH3NH2 • HCI
methylamine

(CH3)2 NH + HCI = (CH3)2 NH • HCI
dimethylamine

(CH3)3 N + HCI = (CH3)3 N • HCI
trimethylamine

The hydrogen atoms of ammonia can also be replaced by groups of
greater complexity, as will be seen below.

Complex ring compounds in which nitrogen forms part of the ring
are termed heterocyclic, such as the alkaloids, purines and some amines,
for instance pyrrolidine (see below).

The plant bases can be conveniently classified into four groups and
this is also to a large extent a natural grouping. They are:

1. Amines )

2. Betaines} Simpler natural bases.

3. Alkaloids.

4. Purine bases.

The first two groups have been termed the simpler natural bases.
They are much more widely distributed in the vegetable kingdom than
the alkaloids and purins, since they have probably much more significance
in general metabolism. The isolation of the simpler bases is a matter of
much greater difficulty than that of the alkaloids: the former are soluble
in water but insoluble in ether and chloroform, and so are not readily
separated from other substances. The alkaloids, however, occur in the
plant as salts of acids and if the plant material is made alkaline the free
bases can be extracted with ether or chloroform.

CH. x] THE PLANT BASES 155

The betaines are amino-acids in which the nitrogen atom is com-
pletely methylated, and, with one or two exceptions, this grouping does
not occur in the true alkaloids. The betaines have only feebly basic
properties.

The alkaloids, in contrast to the simpler natural bases, are rather
restricted in their distribution, many being limited to a few closely
related species or even to one species.

The purine bases are a small group of substances intimately related
to each other and to uric acid.

AMINES.

Methylamine, CH3 . NH2, occurs in the Annual and Perennial Dog's
Mercury (Mercurialis annua and M. perennis) and in the root of the
Sweet Flag (Acorus Calamus).

Trimethylamine, (CH3)3 . N, occurs in leaves of the Stinking Goose-
foot (Chenopodium Vulvaria), in flowers of ,the Hawthorn (Crataegus
Oxyacantha) and Mountain Ash (Pyrus Aucuparia), and in seeds of
Mercurialis annua.

Ptitrescine, NH2 (CH2)4.NH2, occurs in the Thorn Apple (Datura)
and tetramethylputrescine in a species of Henbane (Hyoscyamus
muticus).

Hordenine occurs in germinating Barley grains. It is represented
as:

o'CH2-N(CH3)2

Pyrrolidine is said to occur in small quantities in leaves of the
Carrot (Daucus Carota) and Tobacco (Nicotiana) leaves. It is repre-
sented as :

CH2 - CH2

I ' I
CH2 CM*

Other amines occur among the lower plants (Fungi).
Choline is sometimes classified with the betaines. It is however
intimately connected with the phosphatides (compounds of the fatty
acids with phosphoric acid and nitrogen) which is not the case with the
betaines. It may be represented as :

OH
(CH3)3: N<

XCH2 • CH.2OH

156 THE PLANT BASES [CH.

Choline is very widely distributed in plants. It is a constituent of the
phosphatide, lecithin, and is probably thereby a constituent of all living
cells. It has been found in seeds of the Bean ( Vicia Faba), Pea (Pisum
sativum), Strophanthus, Oat (Avena sativa), Cotton (Gossypium her-
baceum), Beech (Fagus sylvatica), Fenugreek (Trigonella Foenum-
graecum) and Hemp (Cannabis sativa): in seedlings of Lupins, Soy
beans, Barley and Wheat: in Potatoes and Dahlia tubers and in the
subterranean parts of Cabbage (Brassica napus), Artichoke (Helianthus
tuberosus), Scorzonera hispanica, Chicory (Cichorium Intybus), Celery
(Apium graveolens) and Carrot (Daucus Carota) : aerial parts of Meadow
Sage (Salvia pratensis) and Betony (Betonica officinalis), and many
other tissues. It can only be isolated in very small quantity.

BETAINES.

The betaines, as previously stated, are amino-acids in which the
nitrogen atom is completely methylated. Most betaines crystallize with
one molecule of water; thus betaine itself in this condition probably has
the following constitution, from which its relationship to glycine or
aminoacetic acid is indicated:

OH
(CH3)3 : N / H2N • CH2 • COOH

XCH2'COOH

Betaine or hydroxytri methyl- Aminoacetic acid

aminoacetic acid

When dried above 100° C., the betaines lose water and are represented
as cyclic anhydrides; thus betaine becomes:

(CH3)3: N CO

\CH/

The individual betaines, probably on account of their close connexion
with proteins, are more widely distributed than the individual alkaloids.
Further investigation may show an even more general distribution of
betaines.

Betaine or trimethylglycine occurs in all species of Chenopodiaceae
so far examined including the sugar Beet (Beta vulgaris) from which it
derives its name; in some genera only of the Amarantaceae ; in the "Tea
Plant" (Lycium barbarum): in seeds of Cotton (Gossypium herbaceum),
Sunflower (Helianthus annum) and Oat (Avena sativa): in tubers of
Artichoke (Helianthus tuberosus), shoots of Bamboo (Bambusa), leaves
of Tobacco (Nicotiana Tabacum) and in malt and wheat germs.

x] THE PLANT BASES 157

Stachydrine, though a betaine, is included by most writers among
the alkaloids, and this classification has been followed here (see p. 161);
it is probably a derivative of proline (see p. 121).

Betonicine, C7H13O3N, is also, like stachydrine, found in the Be tony
(Betonica officinalis). It is a derivative of oxyproline.

Hypaphorine or trimethyltryptophane, C14H18O2N2, occurs in the
seeds of a tree, Eryihrina Hypaphorus, which is grown for shade in
Coffee plantations.

Trigonelline, like stachydrine, is usually classed with the alkaloids
(see p. 160) but it should probably be included among the betaines on
account both of its structure and of its wide distribution.

Other betaines, trimethylhistidine, ergothioneine, occur in the
Fungi.

ALKALOIDS.

The plant alkaloids, so-called because of their basic properties, have
attracted considerable attention on account both of their medicinal
properties and, in many cases, their intensely poisonous character. They
were also the plant bases to be first investigated. As previously men-
tioned they are not widely distributed, some being, as far as is known,
restricted to one genus, or even species. Moreover, several closely
related alkaloids are frequently found in the same plant. The orders in
which they largely occur are the Apocynaceae, Leguminosae, Papa-
veraceae, Ranunculaceae, Rubiaceae and Solanaceae.

The alkaloids may be present in solution in the cell-sap in the
young tissues, but in older and dead tissues they may occur in the solid
state ; they may be found throughout the plant or more abundantly in
the seed, fruit, root or bark (quinine).

The alkaloids are, as a rule, insoluble in water, but soluble in such
reagents as alcohol, ether, chloroform, etc. The majority are crystalline
solids which are not volatile without decomposition, but a few, for
example coniine, nicotine, which contain no oxygen, are volatile liquids.

The alkaloids occur in the plant as a rule as salts of various organic
acids, such as malic, citric, succinic and oxalic, and sometimes with an
acid peculiar to the alkaloid with which it is united (e.g. quinic acid in
quinine and meconic a6*id in opium). Artificial salts, i.e. sulphates,
chlorides and nitrates, are easily prepared and are readily soluble in
water, and from these solutions the free base is precipitated again on
addition of alkali.

158 THE PLANT BASES [OH.

The alkaloids themselves belong to various classes of compounds,
though the basic character always preponderates. Thus, for example,
piperine is an amide and can be hydrolyzed into the base piperidine
and piperic acid : atropine is an ester made up of the base tropine and
tropic acid.

Various methods are employed for the extraction of alkaloids but
the exact course of events depends on the alkaloid in question. On the
whole the method is either to treat the plant material with alkali and
then extract the free alkaloid with ether or chloroform and finally purify
by making a salt again ; or to extract the alkaloid from the plant with
dilute acid, set free the insoluble, or difficultly soluble, base with alkali,
and then prepare a salt of the base.

Though individual alkaloids have distinctive reactions, the group as
a whole has certain reactions in common, namely the precipitation by
the so-called " alkaloidal reagents." These reagents are tannic, phospho-
tungstic, phosphomolybdic and picric acids, also potassium-mercurio-
iodide solution and iodine in potassium iodide solution.

Expt. 151. General reactions of alkaloids. Make a 0'5 °/0 solution of quinine
sulphate in warm water and add a few drops of each of the following reagents:

(a) Tannic acid solution. A white precipitate is formed.

(6) Mercuric iodide in potassium iodide solution [Brucke's reagent : 50 gms. of
potassium iodide in 500 c.c. water are saturated with mercuric iodide (120 gms.) and
made up to 1 litre]. A white precipitate is formed.

(c) Phosphotungstic acid (50 gms. of phosphotungstic acid and 30 c.c. of cone, sul-
phuric apid are dissolved in water and made up to a litre). A white precipitate is formed.

(d) Iodine in potassium iodide solution. A brown precipitate is formed.

(e) Picric acid solution. A yellow precipitate is formed.

Expt. 152. Extraction of the free base from quinine sulphate. Add strong sodium
carbonate solution drop by drop to some of the quinine sulphate solution until a
white precipitate of quinine is formed. Then add ether and shake up in a separating
funnel. The precipitate will disappear as the quinine passes into solution in the ether.
Separate off the ethereal solution and let it evaporate in a shallow dish. The quinine
is deposited. Take up the quinine again in dilute sulphuric acid and test the solution
with the alkaloidal reagents.

The alkaloids are classified into five groups according to the nucleus
which constitutes the main structure of the molecule. These five groups
are:

1. The pyridine group.

2. The pyrrolidine group.

3. The tropane group.

4. The quinoline group.

5. The isoquinoline group.

THE PLANT BASES

159

Pvridine

Pyrrole

Tropane

Quinoline

Pyrimidine

V

Iminazole

Purine

1. The pyridine alkaloids.

These are, as the name implies, derivatives of pyridine. (Pyridine
is a colourless liquid which boils at 115°C. It is a strong base and
forms salts with acids.)

CH

CH CH

II I
CH CH

N

Pyridine

The more important members of this group are: arecoline, coniine,
nicotine, piperine and trigonelline.

Arecoline occurs in the " Betel Nut " which is the fruit of the Areca
Palm (Areca Catechu).

Coniine occurs in all parts of the Hemlock (Conium maculatum),
but more especially in the seed.

Nicotine occurs in the leaves of the Tobacco plant (Nicotiana
Tabacum). It is a colourless oily liquid which is intensely poisonous.
Its constitution may be represented as :

CH CH2— CH2

S\ II

CH C— CH CH2

I II \/
CH CH N

\X I

N CH3

It is readily soluble in water and organic solvents.

160 THE PLANT BASES [CH.

Expt. 153. Extraction and reactions of nicotine. Weigh out 100 gms. of plug
tobacco and boil up the compressed leaves with water in an evaporating dish or in a
saucepan. Filter off the extract and concentrate on a water-bath. The concentrated
solution is made alkaline with lime and distilled from a round-bottomed flask fitted
with a condenser, the flask being heated on a sand-bath. The distillate has an un-
pleasant smell and contains nicotine in solution. Test the solution with the alkaloidal
reagents employed in Expt. 151. A precipitate will be obtained in each case.

The nicotine can be obtained from solution in the following way. Acidify the
aqueous distillate with oxalic acid and concentrate on a water-bath. Make the con-
centrated solution alkaline with caustic soda, pour into a separating funnel and shake
up with ether. Separate the ethereal extract and distil off the ether. The nicotine
is left behind as an oily liquid which oxidizes in air and turns brown. The alkaloidal
tests should be made again with the extracted nicotine.

Piperine occurs in various species of Pepper (Piper nigrum). The
fruit, which is gathered before it is ripe and dried, yields a black
pepper, but if the cuticle is first removed by maceration, a white pepper.
Piperine is a white solid which is almost insoluble in water but soluble
in ether and alcohol.

Expt. 154. Extraction and reactions of piperine. Weigh out 100 gms. of black
pepper. Put it into an evaporating dish, cover well with lime-water and heat with
constant stirring for 15-20 minutes. Then evaporate the mixture completely to
dryness on a water-bath. Grind up the residue in a mortar, put it into a thimble
and extract with ether in a Soxhlet. Distil off the ether and take up the residue in
hot alcohol from which the piperine will crystallize out. With an alcoholic solution
make the following tests :

(a) Add the alkaloidal reagents mentioned in Expt. 151 and note that a pre-
cipitate is formed in each case.

(6) Pour a little of the solution into water and note that the piperine is pre-
cipitated as a white precipitate.

(c) To a little solid piperine in a white dish add some concentrated sulphuric
acid. It dissolves to form a deep red solution.

Trigonelline occurs in the seeds of the Fenugreek (Trigonella
Foenum-graecum), Pea (Pisum sativum\ Bean (Phaseolus vulgaris),
Strophanthus hispidus, Hemp (Cannabis sativa) and Oat (Avena sativa).
It is also found in the Coffee Bean (Coffea arabica) ; in tubers of
Stachys tuberifera, Potato and Dahlia and in roots of Scorzonera hispanica.
It is really a betaine (see p. 157).

2. The pyrrolidine alkaloids.

These are derivatives of pyrrolidine, of which the mother substance
is pyrrole. (Pyrrolidine is a liquid boiling at 91° C. It is a strong base
and forms stable salts with acids.)

THE PLANT BASES

161

CH— CH

CHo— CH2
II

CH CH CH2 CH2

V XV

NH NH

Pyrrole Pyrrolidine

These alkaloids form a small group containing :

Hygrine and euskhygrine which occur in Coca leaves (Eryihroxylon
Coca).

Stachydrine which occurs in tubers of Stocky s tuverifera and leaves
of the Orange Tree (Citrus Aurantium) and in various other plants

(Betonica). The formula is :

CH2 — CH2

I ' I
CO— CH ^ CH,

\/

-N(CH3)2

from which it is seen that it is really a betaine (see p. 157).

3. The tropane alkaloids.

These are derivatives of tropane, which may be regarded as formed
from condensed piperidine and pyrrolidine groupings. (Tropane is a
liquid boiling at 167JC.)

CH2

/ \
CH2 CH2

I 1

CH CH

\/

NCH3

Tropane

The alkaloids in this group are limited to four natural orders and
are as follows :

Solanaceae : Atropine occurs in the root and other parts of the
Deadly Nightshade (Atropa Belladonna), the Thorn Apple (Datura
Stramonium) and Scopolia japonica. Atropine may be represented as :
CH — O— CO— CH • CHoOH

/ \ I

CH2 CH, C6H5

i I '

CH CH

NCH3
CH2 — CH2

O.

11

102 TIIK PLANT I5ASKS [CM.

Hyosoyamine occurs in the Henbane (Hyoacyamw niger), //.
mutiwui and alno in the Mandrake (Mandragora).

Erythroxylaceae : Cocaine and tropacocalne occur in Coca leaves

(Ki'iithnu'ijln-n CHCII) together wil.h smaller <|iianfifies of allied alkaloids.
( 'ocame has I IK- formula :

H OCOC,,H0

\/
C

/\

CH3 CHCOOCH8

I I

OH CH

\ /

NCH8

OHa— CHa

I'unicaceae: Pollctieritie and «>! her allied alkaloids occur in the root
and slmi of I he I'.mirgranati1 Tivr ( /'/////(•(/ (,' nimiftnn).

Leguininosac : Sparteine occurs in the Broom (Spartium scoparium):
lupinine in I lu \« How and black Lupins (Lupinus luteus and L. niger)
and cytisine in the Laburnum (Cytism Laburnum}.

4. The quinoline alkaloids.

These arc derivatives ol' (jumolinc. ((.^iiinolinc is a colourless liquid

which boils at 239° C.) Its constitution is :

CH CH

CH C CH

I II I

CH C CH

CH N

(Juinolino

Th(\st' alkaloids form l\vo natural i;roii|»s, (u) the cinchona alkaloids,
i.o. (quinine, cmchoninc and allied forms, and (/>) the strychnine alkaloids,
i.e si i vdmme and hnirme.

Quinine occurs in the bark of various species of the genus Cinchona

(Iviihiaceae) which are trees, originally nati\es of S. America, hut now
rultuated on a lar^e scale in (Vylon, ,la\a and India. The species
employed are ( Y. ('(tlistti/tt. An/f/rrm/m, tijficinttliit.surcirubra. The yellow
hark of ( 'ttlistii/tt has the highest pei-ceJita^e, i.e. I*J",,. of alkaloid.

(Quinine is a whit*' solid which cryslalli/es in long needles containing
water of cr\ stalli/.at ion. It is very sligliily soluble in cold water, more
M) in hot but. readily soluble in alcohol, ether and chloroform. With

x] TIIK I'LANT BASKS

acids it form* salts, which are soluble in water, the sulphate being
< -mil UK. iily employed in medicine. Quinine is said to have the following
< (institution :

C,0HJfl(OH)N

OCH:,

Kxpt. 15ft. Extraction «„<! r<>action* of r/uininc. Mix 20 gm,s. of «|ui«-l<lim« with
200 c.c. of water in a basin and than Add KX) gms. of powdered Cinchona bark. St ir
together wdl and then dry the mixture thoroughly on a water-bath, taking care to
|>owdcr the lump-. Tin- dried mixture in then extracted in a Soxhlet. apparatus with
'•lilon.f'onn. Tin- i-hl«.r««f«.nn <-\ti..«i r- th<-n .shaken up in a separating funnel with
25 c.c. of dilute sulphuric acid. The chloroform layer in run of!' and again extracted
v. ill. • ulph uric acid and water extracts are mixed together and n< iitr.,ii/«i

with ammonia. The liquid in evaporated on a w;tt< r kith until uryntalH of quiniin-
.sulj)hate begin to Heparate out. With the quinine milphate th<^ following tentH Mhould
be made. (It in better to tue a solution of the hydrochloride pr« -\> .-m-d i-y adding a
few drop** of hydrochloric acid to the sulphate rotation) :

(a) Test with the alkaloidal reagents of Kxpt. 161.

(6) Add to a little of the solution some bromine water and then Home ammonia.
A green precipitate in formed which gives a green solution with excess of ammonia.

(c) Dissolve a little of the solid quinine sulphate in acetic and ;in«l p«iir into a
large volume of water. A blue opaleaoence is produced which is characteristic of
quinine.

Cinchonine occurs together with quinine in Cinchona bark. It is
very similar in constitution to quinine, the latt« r Ix-ing methoxy-
cinchoniod.

Strychnine and brucine occur in the seeds of Nux Votnica (Strych-
UOH Nux-vomica) and St Ignatius' Bean (8. Ignatii).

Kxpt. 150. Ti»t» for strychnine. Add a little concentrated sulphuric acid to a
small quantity of strychnine in an evaporating dish un<l tin -u add a small amount
of powdered potassium bichromate. A violet coloration is produced which changes
to red and finally yellow.

Curarine, the South American Indian Arrow poison, occurs in
(several species of Stryc/uwtt (/Sf. toxifara and others).

5. The iftoqu'inoline alk<il'.«l

These can be divided into two ^n/up.s: (a) the opium alkaloids and
(/>) the berberine alkaloids.

The opium alkaloids ;«;.;.•! in fall into two classes: (1) tlx- papav-r IIM-

11-2

164 THE PLANT BASES [CH.

group which includes papaverine, laudanosine, narceine, narcotine
and others, and (-2) the morphine group including morphine, apomor-
phine, codeine, thebaine and others.

Opium is the dried latex obtained by making incisions in the cap-
sules of the Opium Poppy (Papaver somniferum).

Allied to the papaverine group is hydrastine which occurs in the
root of Hydrastis canadensis (Ranunculaceae).

The constitution of all these alkaloids is very complex.

Expt. 157. Tests for morphine.

(a) Add a little ferric chloride solution to a solution of a morphine salt. A deep
blue coloration is formed.

(6) Dissolve some morphine in concentrated sulphuric acid and then after
standing about 15 hrs. add concentrated nitric acid. A deep blue- violet colour is
produced which afterwards changes to red.

Berberine occurs in the root of the Barberry (Berberis vulgaris) and
is also found in isolated genera in Anonaceae, Menispermaceae, Papa-
veraceae, Ranunculaceae and Rutaceae.

Corydaline occurs in Corydalis cava (Fumariaceae).

Many other alkaloid substances have been isolated from a large
number of different plants, but since the constitution of most of them
is unknown, they have not been classified.

PURINE BASES.

These substances, as indicated, have a heterocyclic ring structure
and are derivatives of purine : the atoms of the ring are numbered in
the order indicated below:

. | | 1N— CC
HC C — NH !

\ 2C 5C_7N

CH | || \C8

^ 3|S|— 4C— 9(Sr

N — C— N
Purine

Purine itself is a crystalline basic compound (m. p. 211-212° C.)
which forms salts with acids. It is composed of two rings, the pyrimi-
dine and the iminazole: the latter grouping also occurs in histidine
(see p. 121).

x] THE PLANT BASES 165

The chief purine bases which occur in plants are xanthine, caffeine,
theobromine, guanine, hypoxanthine and adenine.

Xanthine may be regarded as 2, 6 -dioxy purine:

HN— C=0

I I

O— C C— NH

\

CH
^

HN— C— N

It is widely distributed in plants and has been found in leaves of the
Tea plant (Thea sinensis), in the sap of the Beetroot (Beta) and in various
seedlings.

Caffeine or theine is 1, 3, 7-trimethylxan thine:

CH3-N— (

I i

O=C C — N ' CH3

CH
CH3'N— C— N

It occurs in the leaves and beans of the Coffee plant (Coffea arabica),
in leaves of the Tea plant (Thea sinensis), in leaves of Ilex paraguensis
(" Paraguay Tea"), in the fruit of Paullinia Cupana and in Kola nuts
(Cola acuminata).

Expt. 158. Preparation of caffeine from tea1. Digest lOOgms. of tea with 500 c.c.
of boiling water for a quarter of an hour. Then filter through thin cloth or fine
muslin using a hot-water filter in order to keep the liquid hot. Wash the residue
with a further 250 c.c. of boiling water. Add to the filtrate a solution of basic lead
acetate until no more precipitate is formed. This removes proteins and tannins.
Filter hot and to the boiling filtrate add dilute sulphuric acid until the lead is pre-
cipitated as sulphate. Filter from the lead sulphate, and concentrate the solution,
with the addition of animal charcoal, to 250-300 c.c. Filter and extract the filtrate
three times with small quantities (50 c.c.) of chloroform. Distil off the chloroform
on a water -bath, and dissolve the residue in a small quantity of hot water. On
allowing the solution to evaporate very slowly, long silky needles of caffeine separate,
which may have a slightly yellow tint, in which case they should be drained, re-
dissolved in water, and boiled with the addition of animal charcoal. The yield
should be about 1/5 gm.

Evaporate a little of the caffeine on a water-bath with bromine water. A reddish-
brown residue is left which becomes purple when treated with ammonia.

1 From Conen, Practical Organic Chemistry.

166

THE PLANT BASES

[CH.

Theobromine is 3, 7-dimethylxanthine:

HN— C=O

I I
O=C C— N'CHg

\
CH

CH3-N— C— N

It occurs in the fruit of the Cocoa plant (Theobroma Cacao), in
leaves of the Tea plant (Thea sinensis) and in the Kola nut (Cola
acuminata).

Guanine and hypoxanthine can be represented respectively as
2-amino, 6-oxypurine and 6-monoxypurine:

HN— C=O HN— C=O

II II

H2N— C C— NH HC C— NH

\

CH

CH

N— C— N
Guanine

N— C— N

Hypoxanthine

They usually occur together and have been found in the germinating
seeds of the Sycamore (Acer pseudoplatanus), Pumpkin (Cucurbita
Pepo), Common Vetch (Vicia sativa), Meadow Clover (Trifolium
pratense), yellow Lupin (Lupinus luteus) and Barley (Hordeum vulgare):
also in the juice of the Beet (Beta).

.

Adenine is 6-aminopurine. It is represented as:

=—

I=C— NH2

I
HC C— NH

CH

N— C — N

It has been found in Beet (Beta), Tea leaves (Thea sinensis) and in
leaves of the Dutch Clover (Trifolium repens).

Guanine, hypoxanthine and adenine are all obtained by the hydro-
lysis of plant nucleoproteins.

THE PLANT BASES 167

REFERENCES
BOOKS

1. Abderhalden, B. Biochemisches Handlexikon, v. Berlin, 1911.

2. Allen's Commercial Organic Analysis. Vegetable Alkaloids (G. Barger),
Vol. 7, 1913, pp. 1-94.

3. Barger, G. The simpler Natural Bases. London, 1914.

4. Henry, T. A. The Plant Alkaloids. London, 1913.

5. Winterstein, B., und Trier, G. Die Alkaloide. Berlin, 1910.

INDEX

Figures in heavy type denote main references.

Abderhalden, 9, 24, 77, 86, 140, 153, 167

Abies pectinata, 136

Acacia, 61

Acacia Senegal, 61

Acer pseudoplatanus, 166

Acetic acid, 79

Acetone, 143, 148

Achroodextrin, 57

Acorus Calamus, 155

Acrolein, 83

Acrylic aldehyde (see Acrolein)

series, 79

Adenine, 166
Adipo-celluloses, 65, 69
Adzuki-bean, 126
Aegopodium Podagraria, 95
Aesculetin, 144, 150
Aesculin, 144, 150
Aesculus, 112, 144, 151

Hippocastanum, 92, 93, 136, 150

Aetiophyllin, 30

Aetioporphyrin, 32

Agar, 13, 49, 50

Alanine, 120

Albumins, 118, 124

Albumoses, 118, 119, 128

Alcoholic fermentation, 21

Alder Buckthorn, 146

Aleurone, 118

Alkaloidal reagents, 124, 158

Alkaloids, 154, 157

Alkanet, 81

Allen, 9, 86, 153, 167

Allium, 62, 70

Cepa, 59, 74

Allocyanidin, 105, 106
Allocyanin, 106, 107
Almond, 61, 80, 126, 145, 147
Aloe, 62, 113

Althaea rosea, 62, 102
Althaein, 102
Alyssum, 110, 111, 112
Amandin, 126
Amarantaceae, 100, 156
Amaranthus, 100
Amines, 154, 155
Amino-acids, 119, 136, 156
Ampelopsidin, 102
Ampelopsin, 102
Ampelopsis quinquefolia, 102
Amphoteric electrolytes, 15, 120
Amygdalase, 145

Amygdalin, 24, 142, 143, 145, 147
Amylodextrin, 57
Amyloid, 66
Ananas sativus, 139

Anchusa qfficinalis, 81

Aniline acetate (test for pentoses), 44

Anonaceae, 164

Anthocyan pigments, 87, 98

artificial, 105

isomerization of, 98

reactions of, 99

Anthocyanidins, 98
Anthocyanins, 98

Antirrhinum, 94

majus, 95, 96, 99
Antiseptics, 18
Apigenin, 95, 144
Apiin, 95, 144
Apiose, 144
Apium graveolens, 156
Apocynaceae, 157
Apomorphine, 164
Apple, 63, 107, 112, 145
Apricot, 126
Aquilegia, 98, 100

vulgaris, 146, 147

Araban, 43, 45, 53, 60, 61
Arabic acid, 61

Arabin (see Gum Arabic)

Arabinose, 41, 43, 44, 53, 60, 61, 62, 63

Arabis, 108, 112

Araceae, 145

Arachidic acid, 79

Arachis hypogaea, 127

Araliaceae, 150

Arbutin, 88, 144, 151

Arctostaphylos Uva-ursi, 151

Areca Catechu, 159

Areca Palm, 159

Arecoline, 159

Arginine, 121, 136

Armstrong, 19, 24, 77, 84, 86, 115, 145, 146,

153
Aromatic acids, 87, 89

alcohols, 87, 88

aldehydes, 87, 88

compounds, 1, 87

Arsenic trisulphide sol, 12, 16
Artichoke, 59, 136, 156
Arum maculatum, 146, 147
Asclepiadaceae, 145

Ash, 81

Asparagin, 136

Asparagus, 59, 60, 88, 143, 151

officinalis, 88

Aspartic acid, 120, 136
Aster, 102

Asterin, 102
Astragalus, 61

gummifer, 61

INDEX

169

Atkins, 77

Atriplex, 100

Atropa Belladonna, 161

Atropine, 161

Anbrietia, 108 . ,

Aucuba, 144

Aucubigenin, 144

Aucubin, 144

Auld, 24, 25

Autolysis, 19, 138, 146

Avena saliva, 125, 126, 156, 160

Avenalin, 126

Bamboo, 137, 156

Bambusa, 137, 156

Banana, 138

Barberry, 164

Barger, 167

Barley, 57, 113, 124, 127, 131, 155, 156,

166

Basidiomycetes, 113
Bassett, 95, 96, 116
Bayliss, 14, 16, 19, 24
Bearberry, 151
Beech, 156

- Copper, 100

-wood, 67

Beet, 63, 71, 72, 76, 100, 107, 136, 137,

138, 156, 165, 166
Behenic acid, 79
Benzaldehyde, 24, 143, 144, 145
Benzidine (test for peroxidase), 109
Benzoic acid, 143
Berberidaceae, 145
Berberine, 164
Berberis vulgaris, 164
Bertholletia excelsa, 81, 83, 125, 126, 135
Beta, 72, 75, 143, 165, 166

- vulgaris, 70, 71, 76, 100, 136, 138,

156

Betaines, 154, 156
Betonica, 161

officinalis, 156, 157
Betonicine, 157
Betony, 156, 157
Betulaceae, 80
Bignoniaceae, 145
Bilberry, 102
Bird Cherry, 45, 61, 145
Bird's-foot Trefoil, 146, 147
Biuret reaction, 122
Blau, 153 .
Blood, 138, 140
Boletus cyanescens,-H3 , .

luridus, 113

Bolton, 116
Boraginaceae, 108
Borneol, 2

Bourquelot, 147, 151, 153
Bran, 45, 53, 54, 55, 68
Bras8ica,*75, 81, 138, 149

alba, 127, 135, 138, 144

campestris, 127, 136

Napus, 80, 156

Brassica nigra, 144, 149
— . oleracea, 136

rapa var. oleifera, 80

Brassidic acid, 79

Brazil nut, 81, 83, 125, 126, 135

Broad Bean, 60, 124, 126, 133, 134, 138,

139, 156
Bromelin, 139
Broom, 162
Brown, 70, 73, 74, 77
Brownian movement, 14
Brucine, 163
Briicke's reagent, 158
Buchner, 21
Buckthorn, 96
Burton, 16
Buttercup, 108
Butter-nut, 126
Butyric acid, 79

Cabbage, 136, 137, 138, 139, 156
Caesalpinia, 91
Caffeic acid, 110
Caffeine, 165
Calanthe, 152
Caldwell, 24, 25
Callistephin, 102
Callistephus chinensis, 102
Calluna erica, 96
Campanulaceae, 58
Campbell, 134, 141
Camphor, 2

tree, 2

Cane-sugar (see Sucrose)
Cannabis sativa, 67, 80, 125, 126, 134, 139,

156, 160
Capparis, 144
Capric acid, 79, 80
Caprifoliaceae, 145, 150
Caproic acid, 79, 80
Caprylic acid, 79, 80
Carbohydrates, 1, 41

in leaves, 69

Carbon assimilation, 5, 26
Carboxylase, 21
Carica Papaya, 139
Carotin, 27, 28, 29, 39
Carrot, 39, 65, 138, 139, 155, 156
Carum, 144

- Petroselinum, 95
Castanea, 91, 93

vulgaris, 92, 126

Castanin, 126

Castor oil, 81

-plant, 80, 83, 84, 125, 136

-seed, 124, 127, 135, 139

Catalase, 20, 21, 23

Catalysts, 17
Catechol, 68, 87, 110, 111
Celastraceae, 145
Celery, 137, 156
Celluloses, 41, 65

reserve, 65

tests for, 66

11—5

170

INDEX

Centaur ea, 144

Cyanus, 98, 100, 101, 102, 104

Centrospermae, 150

Cerasin (see Cherry gum)
Cerasus, 144

Padus, 145

Chaerophyllum sylvestre, 72
Cheiranthus, 106

4 Cheiri, 96, 97, 99, 100, 107,
108

Chenopodiaceae, 100, 156
Chenopodiinn, 136

Vulvaria, 155

Cherry, 45, 61, 63, 80, 102 *
gum, 43, 45, 53

Laurel, 145, 146

Chervil, 72

Chicory, 58, 60, 136, 156
Chittenden, 128, 140
Chlorophyll, 13, 27

a, 27, 29, 32

b, 28, 29, 32
allomerized, 36
colloidal, 35, 37
crystalline, 32

Chlorophyllase, 33
Chlorophyllides, 33
Chlorophyllins, 29
Chodat, 107, 108, 115
Choline, 155
Christmas Eose, 108
Chrysanthemin, 102
Chrysanthemum, 101, 102

indie urn. 102
Chrysin, 95

Cichorium Intybus, 58, 136, 156
Cinchona, 162, 163

Calisaya, 162

Ledgeri'ima, 162

qfficinalis, 162
succirubra, 162

Cinchonine, 163
Citrus, 144

Aurantium, 161

Clark, 76, 78, 115

Clover, 59, 60, 65, 74, 75, 137

Dutch, 166

Meadow, 166

Clupanodonic acid, 80
Coca, 161, 162
Cocaine, 162
Cochlearia, 109

Armoracia, 149

Cocoa, 81

plant, 166

Coconut, 53, 80, 83, 127, 135

• oil, 81

Cocos, 81

mtcifera, 80, 127, 135

Codeine, 164
Co-enzyme, 21

Coffea arabica, 59, 00, 81, 160, 1<J">
Coffee bean, 59, 60, 81, 160, 105
Cold acuminata, 165, 160

Cole, 9, 43, 122
Collodion dialyser, 123
Colloidal state, 4, 10

precipitation of, 15, 16
Columbine, 98, 100, 146, 147
Colza, 80

- oil, 81
Combes, 105, 115
Combretaceae, 150
Compositae, 58, 81, 108, 145, 150
Conglutin, 126, 133
Coniferae, 53, 60, 136, 143, 151
Coniferin, 67, 88, 143, 151
Conifers, 2, 28

Coniferyl alcohol, 88, 143, 151
Coniine, 159
Conium maculatum, 159
Continuous phase, 11, 14
Convoivulaceae, 145
Corchorus, 67
Cork, 69

Cornflower, 98, 100, 101, 102, 104, 106
Corydaline, 164
Corydalis cava, 164
Corylin, 126
Corylus Avellana, 80, 126

var. rubra, 100
Cotton plant, 53, 65, 81

seed, 127, 135, 156

- oil, 81

Cotyledon Umbilicus, 74
Coumarin, 150
Courtauld, 24, 25
Cow Parsnip, 32
Cow Pea, 126
Cradein, 139
Cranberry, 88, 102
Crassulaceae, 108
Crataegus, 108

Oxyacantha, 95, 96, 146, 155
Cresol, 68, 113

Cruciferae, 80, 108, 136, 145, 149
Cucumber, 138, 139
Cucumis Melo, 138

sativus, 138

Cncurbita, 136, 137

maxima, 127

Pepo, 81, 166
Cucurbitaceae, 81, 150
Curarine, 163
Currants, 63

Black, 146

Eed, 146

Cuskhygrine, 161

Cutin, 65

Cuto-celluloses, 65, 69

Cyanidin, 101, 102, 105, 114

Cyanin, 102, 144

Cynips, 91

Cystine, 121, 123

Cytase, 20

Cytisine, 162

Cytisus Laburnum, 162

Czapek, 9, 67, 68, 77

INDEX

171

Dahlia, 58, 75, 102, }13, 136, 137, 138, 156, 160
Dahlia variabilis, 58, 102, 113, 138
Daish, 55, 70, 71, 72, 75, 76, 77
Dandelion, 58, 60, 95, 108
Date-palm, 59, 60
Datura, 155

Stramonium, 161

Daucus Carota, 65, 138, 155, 156

Davis, 42, 55, 70, 71, 72, 75, 76, 77, 78

Dead Nettle, 108

Deadly Nightshade, 161

Dean, 138, 140

Delphmidin, 101, 102, 105, 144

Delphinin, 102, 144

Delphinium, 100, 144

consolida, 97, 102

Dextrin, 41, 56, 57, B9, 70, 73

tests for, 58

Dhurrin, 143, 148
Dialysis, 10, 13
Diastase, 20, 56, 57, 69, 73
Digallic acid, 92
Digitaligenin, 144
Digitalin, 144
Digitalis, 144
Digitalose, 144
Dipsacus, 113
Disaccharides, 41, 51
Dispersed phase, 11, 14 .
Dobson, 71, 76, 78
Dock, 95

Dog's Mercury, 113

annual, 155

perennial, 155

Dunstan, 153

Dyer's Greenweed (Broom), 96
- — Weld (Kocket), 94, 96

Edestin, 125, 126, 128, 134

of Barley, 131

Elaeis guinensis, 80, 81
Elai'dic acid, 79

Elder, 28, 68, 95, 137, 146
Ellagic acid, 90

Emulsin, 20, 88, 142, 145, 147, 151, 152
Emulsions, 11, 12
Emulsoids, 11
Enolic form, 72
- Enzymes, 7, 17

classification of, 19, 20

hydrolysis by, 8, 17

synthesis by, 8, 19

Erepsin, 20, 137
Ergothioneine, 157

Erica cinerea, 94
Ericaceae, 88, 144, 151
Erucic acid, 79
Ervvm Lens, 124, 126, 133
Erythrina Hypaphorus, 157
Erythrodextrin, 57
Erythroxylaceae, 162
Erythroxyhn Coca, 161, 162
Esbach's solution, 124
Essential oils, 2

Ethylene series, 82
Eucalyptus, 91
Euler, 24
Euphorbia, 62
Euphorbiaceae, 80, 145
Everest, 98, 105, 115, 116
Ewart, 115
Excelsin, 126, 135

Fagus sylvatica, 156

var. purpurea, 100

Fats, 1, 79

— tests for, 81
Fatty acids, 79

synthesis of, 85

Fehling's test, 49
Fenugreek, 156, 160
Ferric hydroxide sol, 12
Fibrin, 139

carmine, 140

Fichtenholz, 151, 153

Ficus, 139

Fig, 139

Fischer, 92, 115

Fisetin, 97, 105

Flavone pigments, 87, 93, 104

Flavonol pigments, 87, 93, 104

Flax, 62, 67, 80, 125, 127, 147, 148

Flowering Currant, 93

Forget-me-not, 108

Formaldehyde, 26, 27, 36, 38

Fraxetin, 144

Fraxin, 144

Fraxinus, 144

excelsior, 81

Freudenberg, 92, 115
Fructomannans, 59, 69
Fructose (see Laevulose)
Fumariaceae, 164

Fungi, 20, 113, 155, 157 —
Funkia sinensis, 74
Furfural, 44, 67

phloroglucide, 55, 64

Gaillardia, 101

Galactans, 41, 49, 60, 61, 69

Galactoaraban, 60, 69

Galactomannan, 59, 60, 69

Galactose, 41, 47, 49, 50, 60, 61, 62, 63, 144

Galactoxylan, 60, 69

Galanthus, 55

nival is, 70, 95
Galeopsis, 33

Tetrahit. 32

Gallic acid, 89, 90
Gall-nuts, 89
Gallotannic acid, 92
Galls, 91

Oak, 92

Garden Cress, 80
Gaultheria, 144
Gaultherin, 144
Gelatine, 13
Gels, 13, 63

172

INDEX

Genista tinctoria, 96
Gliadin, 118, 127, 130
Globulins, 118, 124
Glucomannans, 59, 69
Glucose, 41, 45, 46, 56, 62, 70, 73, 142
a and /3, 48

tests for, 48

Glucosides, 48, 72, 87, 91, 142

a and £, 48, 143

coumarin, 150

cyanophoric, 145

mustard-oil, 149

Glucotropaeolin, 144
Glutamine, 136
Glutaminic acid, 120, 136
Glutelins, 118, 127, 130
Gluten, 131

Glutenin, 130
Glyceria aquatica, 146
Glycerol, 79, 82, 83
Glycine, 120, 156
Glycine hixpid-a, 126
Glycinin, 126
Glycogen, 21
Glycogenase, 21, 24
Glyoxylic reaction, 122
Gold sol, 12, 16
Goodeniaceae, 58
Gooseberry, 63, 65, 146
Goosefoot, 136

Stinking, 155

Gosney, 86
Gossypium, 53, 81, 144

herbaceum, 65, 81, 127, 135, 156

Goutweed, 95

Graham, 13

Graminaceae, 80, 130, 145, 150

Grape, 102

sugar (see Glucose)

Great Millet, 148
Greengage, 112
Greshoff, 145, 153
Guaiacol, 68
Guaiaconic acid, 107
Guaiacum gum, 107
Guaiacum qfficinale, 107

sanctum, 107

Guanine, 166
Guignard, 149, 153
Gulose, 46

Gum Arabic, 12, 43, 53< 61
Gum Tragacantu, 61
Gums, 41, 49, 60
Gun-cotton, 66
Guttiferae, 150

Haas, 9

Harden, 22, 23, 24, 25

Harris, 134, 141

Hatschek, 16

Hawthorn, 95, 96, 108, 146, 155

Haynes, 63, 78

Hazel, 80

-nut, 126

Hazel, red-leaved, 100
Hedge Woundwort, 32
Helianthus, 60, 75, 136

annuus, 55, 74, 81, 127, 135,

156

tuberosus, 58, 59, 74, 136, 156

Helleborus niger, 108

Hemerocallis fulva, 74
Hemi-cellulose, 60, 69
Hemlock, 159
Hemp, 67, 80, 125, 126, 156, 160

- seed, 134, 139
Hemp-nettle, 32
Henbane, 155, 162
Henry, 24, 25, 153, 167
Heracleum, 33

Sphandylium, 32
Hesperidin, 144
Hesperitin, 144
Hexoses, 41, 45
Hill, 9
Histidine, 121, 137

trimethyl, 157

Hollyhock, 62, 102

Hop, 74

Hopkins, 122

Hordein, 127, 131

Hordenine, 155

Hordeum vulgare, 57, 74, 113, 124, 127, 131,

166

Horse Chestnut, 92, 93, 112, 136, 150
Horse-radish, 109, 110, 149
Horsfall, 96, 116
Horton, 145, 153
Hummel, 96, 97, 116
Humulus Lupulus, 74
Hyacinth, 138, 139
Hyacinthus, 55, 58

orientalis, 138

Hydrastine, 164
Hydrastis canadensis, 164
Hydrocharis Morsus-ranae, 74
Hydroquinone, 87, 88, 144, 151
Hygrine, 161

Hymenophyllum demissum, 74
Hyoscyamine, 162
Hyoscyamus muticus, 155, 162

— niger, 162
Hypaphorine, 157
Hypoxanthine, 166

Idaein, 102

Idose, 46

Ilex paraguensis, 165

Iminazole, 159, 164

Indican, 115, 144, 152

Indigo, 113, 115, 152

plants, 152

Indigofera, 113, 144

Anil, 152

erecta, 152

sumatrana, 152

tinctoria, 152

Indol, 68

INDEX

173

Indoxyl, 115, 144, 152
Inulase, 20, 58
Inulin, 41, 50, 58

tests for, 59

Invertase, 20, 21, 24, 50, 76
Invert sugar, 50

Iris, 55, 58
Irvine, 71, 76, 78
Isatis tinctoria, 113, 152
Isochlorophyllins, 30
Isoleucine, 120, 136
Isolinolenic acid, 80
Iso-oleic acid, 79
Isoquercitrin, 144
Isoquinoline, 159
Isothiocyanate, acrinyl, 144

allyl, 144, 149

benzyl, 144

p-hydroxybenzyl, 149

Jacobinia, 113
Japanese lacquer, 113
Jasminum, 143
Jerusalem Artichoke, 58
Jorgensen, 27, 36, 40
Juglandaceae, 80
Juglans cinerea, 126

nigra, 126

regia, 80, 92, 126

Juglansin, 126

Jute, 67

Kaempferol, 97, 105, 144

Kastle, 76, 78

Keeble, 115

Keracyanin. 102

Kidd, 27, 36, 40

Kidney Bean, 60, 124, 126, 133, 134, 136,

160

Kishida, 93, 116
Kola nut, 165, 166

Labiatae, 108
Laburnum, 162
Laccases, 112
7-Lactone, 47
Laevulose, 41, 50, 58, 70

tests for, 51

Lamium album, 108

Larch, 92

Larix europaea, 92

Larkspur, 97, 100, 102

Latex, 11

Lathy rus odoratus, 74

pratensis, 74

Laudanosine, 164

Laurie acid, 79, 80

Laurocerasin (see Prulaurasin)

Leathes, 86

Lecythidaceae, 81, 83, 150

Legumelin, 124, 134

Legumin, 125, 126, 133

Leguminosae, 59, 74, 125, 133, 145, 150,

157, 162

Lemon, 2

Lentil, 124, 126, 133, 134

Lepidium, 144

sativum, 80

Leucine, 120, 136
Leucosin, 23, 124, 130
Lignin, 65, 67, 68
Ligno-celluloses, 65, 67
Lignon (see Lignin)
Ligustrum, 143
Lilac, 65, 95
Liliaceae, 150
Lilium bulbiferum, 60

candidum, 60, 95

Martagon, 60

Lily, 60

White, 95

Lima-bean, 126
Limonene, 2
Linaceae, 80, 145
Linamarin, 143, 148
Ling, 94, 96
Linolenic acid, 80
Linolic acid, 80
Linseed, 62, 81, 125, 135
Linum, 62, 143, 148

perenne, 147

usitatissimum, 67, 80, 81, 125, 127,

135

Lipase, 20, 84
Lobeliaceae, 58
Loganiaceae, 150
Lotase, 148
Lotoflavin, 144, 148
Lotus, 144

arabicm, 148

corniculatus, 74, 146, 147

uliginosus, 146

Lotusin, 144, 148
Lubrzynska, 86
Lucerne, 65

Lupin, 60, 126, 133, 136, 138, 139, 156, 162,

166

Lupinine, 162
Lupinus, 60, 74, 126, 136, 137

albus, 137

hirsutus, 138

luteus, 133, 137, 162, 166

niger, 162

Luteolin, 96, 105
Lycium barbarum, 156
Lycopersicum esculentwn, 138
^Lysine, 121, 137

Lyxose, 43

Mackenzie, 77

Magnoliaceae, 150

Maize, 80, 126, 127, 132, 138, 139

cobs, 53

Mallison, 116, 117
Mallow, 102
Malt, 76

Maltase, 19, 20, 21, 22, 41, 75
Maltose, 19, 51, 56, 57, 69, 70, 73, 75

174

INDEX

Maltose, tests for, 52
Malva, 144

- sylvestris, 102
Malvaceae, 81
Malvidin, 102, 144
Malvin, 102, 144

Mandelonitrile glucoside (see Prunasin)
Mandragora, 162
Mandrake, 162
Mangold, 70, 71, 72, 75
Mangrove, 91
Mannans, 41, 50, 59, 69
Mannocelluloses, 59
Mannose, 41, 46, 50, 59, 62, 144
Martin, 117
Matthiola, 95, 108
Maxwell, 60, 78
Maysin, 126
Meadow-Bue, 146

-Sage, 156

-Sweet, 88, 152

Medicago sativa, 65
Mekocyanin, 102
Melon, 138, 139
Mendel, 128, 140
Menispermaceae, 164
Menthol, 2
Mercerised cotton, 66
Mercurialis annua, 113, 155

perennis, 113, 155

Metaproteins, 118, 127

reactions of, 128
Methylamine, 155
Methylene blue, 23
Methyl salicylate, 144
Mieg, 117
Mignonette, 96
Milk, 11
Miller, 86

Millon's reaction, 122
Mirande, 146, 153
Mistletoe, 62
Molisch's reaction, 123
Monocotyledons, 55, 58, 70
Monosaccharides, 41, 42
Moore, 115
Moore's test, 48
Moraceae, 80
Morphine, 164
Morris, 70, 73, 74, 77
Mountain Ash, 145, 155
Mucic acid, 49, 50, 62, 63
Mucilages, 41, 49, 50, 60, 62, 65
Musa sapientum, 138
Muscari, 58
Mustard, Black, 80, 149

White, 80, 127, 149

seed, 135, 138

Myosotis, 108
Myrica, 144
Myricetin, 97, 105, 144
Myricitrin, 144
Myristic acid, 79, 80
Myrosin, 20, 142, 149

Myrtaceae, 145, 150
Myrtillidin, 102, 103
Myrtillin, 102

Nagai, 93, 116

a-Naphthol tests, 44, 68, 109
Narceine, 164
Narcissus, 95, 96, 106

incomparabilis, 96

poeticus, 95

Tazetta, 96

Narcotine, 164
Narigenin, 144
Naringin, 144

Nasturtium, 55,f60, 75, 101, 138

Garden, 70, 72, 136

Nepenthes, 139

Nettle, 28
Neville, 62, 78
Newbury, 116
Nicotiana, 155

— Tabacum, 156 159
Nicotine, 159, 160
Nolan, 117
Norris, 23, 25
Nucleic acid, 127
Nucleoproteins, 118, 127, 166
Nux Vomica, 163

Oak, 91, 93, 96

- wood, 67, 92
Oat, 125, 126, 156, 160
Oenidin, 102, 103, 144
Oenin, 102, 144

Oil Palm, 80
Olea europaea, 81
Oleaceae, 81, 145, 150
Oleic acid, 79, 80
Olive, 81

oil, 81

Onion, 59, 96, 97, 108
Onslow, 110, 111, 115
Opium, 164

- Poppy, 80, 164
Ora'che, 100
Orange Tree, 161
Orchid, 89, 113, 152
Orchidaceae, 60
Orchis Morio, 62
Orcinol, 68

test for pentoses, 44

Ormerod, 86

Oryza sativa, 127

Osazones, 49

Osborne, 130, 131, 132, 134, 135, 140

Osrnic acid, 81

Ostwald, 13

Oxidases, 107, 108, 151, 152

Oxidizing enzymes, 8, 107

Oxybenzoic acid, 144

Oxyproline, 157

Paeonia, 60, 85, 144

officinalis, 93, 99, 102, 103

INDEX

175

Paeony, 60, 93, 99, 102, 103
Palladin, 9, 114, 115
Palm, 59, 60

oil, 81

Palmaceae, 80
Palmitic acid, 79, 80
Pansy, 96, 102
Papain, 139
Papaver orientale, 113

Rhoeas, 102

somniferum, 80, 164

Papaveraceae, 80, 157, 164
Papaverine, 164

Papaw Tree, 139

Parkin, 60, 70, 78

Parsley, 95

Passifloraceae, 145

Paullinia Cupana, 165

Pea, 57, 60, 72, 74, 75, 108, 122, 124,

125, 126, 133, 134, 136, 138, 139, 156,

160

Peach, 80, 126, 145
Pea-nut, 127

Pear, 107, 110, 111, 112, 151
Pectase, 20, 65
Pectic substances, 41, 63, 69
Pectin, 63, 64
Pectinase, 20
Pectinogen, 63, 64
Pectocelluloses, 65, 69
Pelargonidin, 101, 102, 105, 144
Pelargonin, 102, 103, 144
Pelargonium, 144

zonale, 93, 102, 103, 138
Pelletierine, 162

Pentosans, 42, 63, 54, 55, 61, 67, 69, 70
Pentoses, 41, 42, 54, 55, 63, 64, 70
Peonidin, 102, 144
Peonin, 102, 144
Pepper, 160
Peppermint, 2
Pepsin, 20, 128, 137
Peptones, 118, 119, 128, 129, 137
Perkin, 95, 96, 97, 115, 116
Peroxidase, 20, 21, 22, 107, 108, 109

inhibitor, 23

Peroxides, 108
Petunia violacea, 102
Petunidin, 102
Petunin, 102
Phaeophorbides, 34
Phaeophytin, 31, 37, 38
Phajus, 113, 152
Phaselin, 124, 134
Phaseolin, 126, 133
Phaseolunatin, 148
Phaseolus, 60, 136, 143

lunatus, 126, 148

multiflorus, 74, 138, 139

radiatus, 126

vulgaris, 124, 126, 133, 134, 137,

160

Phenol, 68
Phenols, 87

Phenylalanine, 121, 122, 137
p-Phenylenediamine (test for peroxidase),

109

Philip, 16
Phipps, 97, 116
Phloretin, 144
Phloridzin, 144
Phloroglucin, 7, 68, 88

(test for pentoses), 44
Phlox, 95
Phoenix, 60
Phospha tides, 155
Phosphotungstic acid, 124, 158
Phyllins, 30

Phytelephas macrocarpa, 60
Phytochlorins, 31
Phytol, 30, 33, 38
Phytolacca, 100
Phytolaccaceae, 100
Phytorhodins, 31
Picea excelsa, 136
Picramic acid, 146
Pine-apple, 139
Pinene, 2
Pink, 95

Pinus sylvestris, 136
Piper nigrum, 160
Piperaceae, 150
Piperidine, 161
Piperine, 160
Pisum, 60, 122, 136, 137
sativum, 57, 72, 74, 75, 108, 124,

126, 133, 134, 138, 139, 156, 160
Pitcher-plant, 139
Pittosporaceae, 150
Plantago, 144

lanceolata, 95

Plastid pigments, 39, 101
Plimmer, 9

Plum, 61, 80, 107, 126, 145
Polarization, 47
Polemoniaceae, 150
Polygalaceae, 150
Polygonum, 144

tinctorium, 152

Polypeptides, 118

Polysaccharides, 41, 53

Pomegranate Tree, 162

Poplar, 95, 152

Poppy, 102

Populin, 143

Populus, 74, 95, 143, 152

Porphyrins, 32

Portulaca, 100

Portulacaceae, 100

Potassium hydrogen sulphate, 144

Potato, 70, 75, 110, 111, 112, 113, 114, 126,

136, 137, 156, 160
Priestley, 36, 40
Primulaceae, 150
Prolamins, 118, 127, 130
Proline, 121, 137, 157
Proteaceae, 150
Proteases, 23, 137

176

INDEX

Proteins, 2, 12, 16, 118

crystalline, 125, 127, 134

of cereals, 130

of fat-containing seeds, 134

of Leguminosae, 133

tests for, 122

Proteoses, 118, 128
Protocatechuic acid, 89, 90, 110
Protoplasm, 4, 8
Prulaurasin, 144, 146
Prunase, 145

Prunasin, 144, 145
Prunus, 97, 143, 144, 145

Amygdalus, 61, 80, 126, 145

Armeniaca, 126

Cerasus, 45, 61, 80, 102

domestica, 61, 80, 126, 145

Laurocerasus, 145, 146

Padus, 45, 61

Persica, 80, 126, 145

Prussic acid, 24, 143, 144, 145
Punica Granatum, 162
Punicaceae, 162
Pumpkin, 81, 166

Purine, 159, 164

- bases, 127, 154, 164
Purpurogallin, 110
Putrescine, 155
Pyridine, 159
Pyrimidine, 159, 164
Pyrogallol, 68, 109, 110
Pyrola, 151
Pyrrole, 159, 161
Pyrrolidine, 155, 161
Pyrus, 143

- Aucuparia, 145, 155

communis, 151

Mains, 145

Pyruvic acid, 86

Quercetin, 96, 105, 106, 144
Quercitrin, 144
Quercus, 91, 96, 144

Robur, 93
Quinine, 158, 162
Quinoline, 159, 162

Radish, 127

Eaffinose, 41

Eanunculaceae, 145, 150, 157, 164

Ranunculus acris, 108

aquatilis, 136

Eape, 80, 127

Raphanus sativus, 127

Easpberry, 65

Eed Seaweeds, 49

Eeductase, 20, 21, 23

Eeed Poa, 146

Eeeves, 141

Reseda luteola, 94, 96
JEeserve celluloses, 65
U materials, 9, 55, 58, 71, 80, 118

Eesorcinol, 68, 87

Eespiration, 5, 71, 114

Eespiration pigments, 114
Eeversible reactions, 18
Eeynolds Green, 84, 86
Ehamnaceae, 145, 150
Rhamnose, 96, 144
Rhamnus, 96

Frangula, 146
Rhizophora, 91
Ehodophyceae, 13, 49
Ehubarb, 63
Rhus, 91, 97

Coriaria, 92

Cotinus, 93

vernicifera, 113

Ribes Grossularia, 146

— nigrum, 146

- rubrum, 146

- sanguineum, 93
Bibose, 43

Eibwort Plantain, 95
Eice, 127

Eicin, 124, 135
Eicinoleic acid, 80
Ricinus, 81, 83, 85, 136

communis, 80, 84, 124, 125, 127,
135, 139

Eobertson, 71, 76, 78
Robinia, 144
Eobinin, 144
Rosa, 144

gallica, 102, 104

Eosaceae, 61, 80, 144, 145, 150
Eose, 93, 99

Eosemary, 2

Eubiaceae, 60, 81, 113, 145, 157, 162

Rumex obtusifolius, 95

Ruscus, 59

Russula nigricam, 113

Ruta, 144

Eutaceae, 145, 150, 164

Eutin, 144

Eye, 124, 127, 132

Saccharomyces, 20
St Ignatius' Bean, 163
Salicin, 48, 88, 143, 148, 152
Salicylic acid, 89

alcohol, 48, 88, 148, 152

aldehyde, 88

Saligenin, 88, 143, 148, 152
Salix, 88, 143, 152
Salkowski, 53, 78
Salvia pratensis, 156
Sambucus, 137, 144

nigra, 68, 95, 146
Sambunigrin, 144, 146
Saponaria, 150
Saponification, 82
Saponins, 11, 150
Sawdust, 45, 54

Sawyer, 42, 55, 70, 71, 72, 76, 77
Saxifragaceae, 145, 150
Scarlet Geranium, 93, 102, 103, 138

Eunner, 138, 139

INDEX

177

Schenckia blumenaviana, 113
Schryver, 38, 39, 40, 63, 78
Schulze, 60, 78
Schweizer's reagent, 66
Scilla, 58, 62, 70
Scopolia japonica, 161
Scorzonera, 143

— hitpanica, 156, 160
Scotch Fir, 136
Secale cereale, 124, 127, 132
Seliwanoff's test, 51
Serine, 120
Shibata, 93, 116
Silicic acid, 13
Silver Fir, 136
Silver sol, 12, 16
Sinalbin, 144, 149
Sinapin acid sulphate, 144, 149
Sinapis alba, 80, 149

nigra, 80

Sinigrin, 142, 144, 149
Skatol, 68

S medley, 86
Snapdragon, 95, 96, 99
Snowdrop, 55, 70, 95
Soap, 11, 12, 16, 82, 83

tests for, 83

wort, 150

Sodium picrate test, 146
Solanaceae, 157, 161
Solanum, 75

tuberosum, 70, 113, 126

Sorghum, 143

vulgare, 148

Soy-bean, 126, 156

Spanish (Sweet) Chestnut, 91, 92, 93, 126

Sparteine, 162

Spartium scoparium, 162

Spatzier, 149, 153

Spinach, 138

Spiiiacia, 138

Spiraea, 144

Vlmaria, 88, 152

Spruce Fir, 136
Squash, 127
Stachydrine, 157, 161
Stachys sylvatica, 32

tuberifera, 160, 161
Starch, 12, 16", 55, 69

soluble, 56, 74

tests for, 56

Stearic acid, 79 *
Steiger, 60, 78
Sterculiaceae, 81
Stereoisomerism, 43, 46, 143
Stiles, 27, 40

Stock, 95, 108
Stoll, 27, 40, 110, 117
Straw, 45, 53, 54, 68
Strawberry, 63, 65
Strophanthidin, 144
Strophanthin, 144
Strophanthus, 144, 156

hispidus, 160

Strychnine, 163
Strychnos Ignatii, 163

Nux-vomica, 163

toxifera, 163

Substrate, 18
Sucrose, 41, 52, 70, 76

tests for, 52

Sulphur reaction (for proteins), 123

Sumac, 91, 92, 97

Sunflower, 55, 75, 81, 127, 135, 136, 156

Suspensions, 11, 12

Suspensoids. 11

Sweet Flag, 155

Sycamore, 166

Synthesis by condensation, 3

of aromatics, 6

of carbohydrates, 5, 26

of fats, 6, 85

of proteins, 6, 119

Syringa, 143

vulgaris, 65, 74, 95
Syringenin, 143
Syringin, 143

Talose, 47

Tannic acid, 75, 92, 123, 158

Tannin, 73, 74, 89, 90, 111

- reactions of, 91
Taraxacum, 60

ojficinale, 58, 95, 108

Tautomerism, 48

Taylor, 16
Tea, 89, 92, 165

plant, 165, 166

Teasel, 113

Terpenes, 2

Thalictrum aquilegifolium, 146

Thea, 144

— sinensis, 165, 166
Thebaine, 164
Theine (see Caffeine)
Theobroma Cacao, 81, 166
Theobromine, 166
Thorn Apple, 155, 161
Thyme, 2

Thynielaeaceae, 150
Thymol, 68
Tiglic acid, 79
Tiliaceae, 145,
Tobacco, 15£
Tomato, 138
Tragacanth, 61
Trier, 167
Trifolium, 59, 137

ochroleucum, 74

pratense, 65, 74, 75, 166

repens, 166

Trigonella Foenum- grace um, 156, 160-
Trigonelline, 157, 160
Trimethylamine, 155
Trisaccharides, 41
Tristearin, 82
Triticum, 113

vulgare, 124, 127, 130, 13$

178

INDEX

Trommer's test, 49

Tropacocaine, 162

Tropaeolum, 60, 75, 136, 137, 144

majiu, 55, 70, 72, 74, 101,

138

Tropane, 159, 161
Trypsin, 139
Tryptophane, 23, 121, 122, 137, 138

trimethyl, 157
Tuberin, 126
Tulip, 138, 139
Tulipa, 138

Turnip, 63, 65, 75, 136, 137, 138
Tyrosinase, 113, 114
Tyrosine, 113, 121, 122, 137

Ultramicroscope, 14
Umbelliferae, 60, 108
Urtica, 28
Urticaceae, 145
Usher, 36, 40

Vaccinium, 151

Myrtillus, 102

Vitis-Idaea, 88, 102

Valine, 120, 136

Van Eijn, 153
Vanilla planifolia, 89
Vanillin, 67, 89
Vegetable ivory, 60

— — marrow, 136
Vernon, 24
Vetch, 124, 126, 133, 134, 136, 148

common, 146, 166

- hairy, 146
Vicia, 136, 137, 144

- angustifolia, 148

Faba, 124, 126, 133, 138, 139, 156

- hirsuta, 74, 146

saliva, 74, 124, 126, 133, 136, 137,

146, 166

Vicianin, 144, 148
Vicianose, 144, 148
Vicilin, 126, 133
Vigna sinensis, 126
Vignin, 126
Vine, 70

Vines, 138, 139, 141
Viola, 108

odorata, 55, 95, 99

Viola tricolor, 96, 102
Violaceae, 58
Violanin, 102
Violet, 55, 95, 99, 108
Virginian Creeper, 102
Viscum album, 62
Vitis, 144

vinifera, 70, 102

Voorhees, 130, 141

Waage, 88, 116

Wallflower, 96, 97, 99, 100, 101, 107, 108
Walnut, 53, 80, 92, 126
American, 126
Walther, 113, 116
Water Kanunculus, 136
Wax, 69
Weil, 117

Wheat, 56, 113, 124, 127, 130, 139, 156
Wheldale, 94, 95, 96, 98, 104, 115, 116
Whitley, 115
Wig Tree, 93
Wilkinson, 97, 116
Willow, 48, 88, 152
Willstatter, 27, 40, 98, 101, 102, 106, 110,

116, 117
Wine, 89

Winterstein, 153, 167
Woad, 113, 152
Wohlgemuth, 24
Wood Gum, 53

Xanthine, 165

Xanthone, 87

Xanthophyll, 27, 28, 29, 39

Xanthoproteic reaction, 122

Xylan, 45, 53, 54, 55, 60, 67

Xylonic acid, 45, 54

Xylose, 41, 43, 45, 53, 54, 60, 61, 62, 63

Yeast, 20

Zea Mays, 80, 126, 127, 132, 138
Zechineister, 102, 117
Zein, 127, 132
Zilva, 22, 23, 24, 25
Zollinger, 117
Zymase, 20, 21, 22
Zymin, 21, 22
Zymogen, 84

CAMBRIDGE : PRINTED BY J. B. PEACE, M.A., AT THE UNIVERSITY PRESS

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