group. For this reason, they very easily unite together, in the same way
that all acids and bases unite, to form larger molecules, the linkage
taking place between the basic NH_{2} group of one molecule and the acid
COOH group of the other, as indicated by the following equation:
R R R R
| ---------- | | |
HOOC·C·N-|H + HO|OC·C·NH_{2} = HOOC·C·N-OC·C·NH_{2} + H_{2}O
| | ---------- | | | |
H H H H H H
It is obvious that the compound thus formed still contains a free NH_{2}
group and a free COOH group, and is, therefore, capable of linking to
another amino-acid molecule in exactly the same way; and so on
indefinitely. In actual laboratory experiments, as many as eighteen of
these amino-acid units have been caused to unite together in this way, and
the resulting compounds thus artificially prepared have been found to
possess the characteristic properties of natural proteins.
These artificially prepared, protein-like, substances have been called
"polypeptides," and the individual amino-acids which unite together to form
them are called "peptides." Thus, a compound which contains three such
units linked together is called a "tripeptid"; one which contains four, a
"tetrapeptid." The use of the term "peptid" was suggested by the fact that
these amino-acids are produced from the hydrolysis of proteins by the
digestive enzyme _pepsin_.
The peptid units of any such complex as those which have been referred to
in the preceding paragraphs may be linked together in a great variety of
ways. Thus, in a tetrapeptid containing units which may be designated by
the letters _a_, _b_, _c_, and _d_, the arrangement may be in the orders
_abcd_, _bacd_, _acbd_, _dbca_, etc., etc. Similarly, the same peptid unit
may appear in the molecule in two or more different places. Hence, the
number of possible combinations of amino-acids into protein molecules is
very great. Further, it is possible that the peptid units in natural
proteins may be united together through other linkages than the one
illustrated above, as they often contain alcoholic OH groups in addition to
the basic NH_{2} groups, and these OH groups may form ester-linkages with
the acid (COOH) groups of other units. Still other acid and basic groups
are present in some of the amino-acids which have been found in natural
proteins, so that the possibility of variation in the polypeptid linkages
is almost limitless.
INDIVIDUAL AMINO-ACIDS FROM PROTEINS
About twenty different amino-acids have been isolated from the products of
hydrolysis of natural proteins, and this number is being added to from time
to time, as the methods of isolation and identification of these compounds
are improved. Many of these same amino-acids have been found in free form
in plant tissues, particularly in rapidly growing buds, or shoots, or in
germinating seeds, where they undoubtedly exist as intermediate products in
the transformation of proteins into other types of compounds.
These amino-acids, grouped according to the characteristic groups which
they contain, are as follows:
A. Monoamino-monocarboxylic acids:
Glycine, C_{2}H_{5}NO_{2}, CH_{2}NH_{2}·COOH, amino-acetic acid.
Alanine, C_{3}H_{7}NO_{2}, CH_{3}·CHNH_{2}·COOH, amino-propionic acid.
Serine, C_{3}H_{7}NO_{3}, CH_{2}OH·CHNH_{2}·COOH, oxy-amino-propionic
acid.
CH_{3}
\
Valine, C_{5}H_{11}NO_{2}, CH·CHNH_{2}·COOH,
/
CH_{3}
amino-isovalerianic acid.
CH_{3}
\
Leucine, C_{6}H_{13}NO_{2}, CH·CH_{2}·CHNH_{2}·COOH,
/
CH_{3}
amino-isocaproic acid.
CH_{3}
\
Isoleucine, C_{6}H_{13}NO_{2}, CH·CHNH_{2}·COOH,
/
C_{2}H_{5}
amino-methylethyl-propionic acid.
/ \
Phenylalanine, C_{9}H_{11}NO_{2}, | |CH_{2}·CHNH_{2}·COOH,
| | phenyl-amino-propionic acid.
\ /
/ \
Tyrosine, C_{9}H_{11}NO_{3}, | |CH_{2}·CHNH_{2}·COOH,
OH| | paraoxy-phenylalanine.
\ /
Cystine, C_{6}H_{12}N_{2}O_{4}S_{2}, HOOC·CHNH_{2}·CH_{2}S-SH_{2}C·
CHNH_{2}·COOH, di(thio-amino-propionic acid).
B. Monoamino-dicarboxylic acids:
Aspartic acid, C_{4}H_{7}NO_{4}, HOOC·CH_{2}·CHNH_{2}·COOH,
amino-succinic acid.
Glutamic acid, C_{5}H_{9}NO_{4}, HOOC·CH_{2}·CH_{2}·CHNH_{2}·COOH,
amino-glutaric acid.
C. Diamino-monocarboxylic acids:
Ornithine, C_{5}H_{12}N_{2}O_{2},
H_{2}N·CH_{2}·CH_{2}·CH_{2}·CHNH_{2}·COOH, di-amino-valerianic acid.
Lysine, C_{6}H_{14}N_{2}O_{2},
H_{2}N·CH_{2}·CH_{2}·CH_{2}·CH_{2}·CHNH_{2}·COOH,
di-amino-caproic acid.
Arginine, C_{6}H_{14}N_{4}O_{2},
NH_{2}
/
HN=C
\
NH·CH_{2}·CH_{2}·CH_{2}·CHNH_{2}·COOH,
guanidine-amino-valerianic acid.
Di-amino-oxysebacic acid, C_{11}H_{12}N_{2}O_{3}.
Di-amino-trioxydodecanic acid, C_{12}H_{26}N_{2}O_{3}.
D. Monoimido-monocarboxylic acids:
Proline, C_{5}H_{9}NO_{2}, H_{2}C---CH_{2}
| |
H_{2}C CH·COOH, pyrrolidine-carboxylic
\ / acid
N
|
H
Oxyproline, C_{5}H_{9}NO_{3}, proline with one (OH) group.
E. Monoimido-monoamino-monocarboxylic acids.
Histidine, C_{6}H_{9}N_{3}O_{2}, HC===C--CH_{2}·CHNH_{2}·COOH,
| | imidazole-amino-propionic
N NH acid.
\\/
C
|
H
Tryptophane, C_{11}H_{12}N_{2}O_{2},
/ \
| |---C--CH_{2}·CHNH_{2}·COOH, indole-amino-propionic acid.
| | ¦
| | CH
\ /\ /
N
|
H
As has been said, other amino-acids are being found, from time to time, as
additional proteins are examined, or as better methods of examination of
the cleavage products of the natural proteins are devised.
COMPOSITION OF PLANT PROTEINS
The distribution of the different amino-acids in some of the different
plant proteins which have been examined in this way is shown in the
following table:
Letter code for column headings:
A = Gliadin (wheat).
B = Hordein (barley seed).
C = Zein (corn).
D = Legumin (vetch).
E = Edestin (hemp).
F = Globulin (squash seed).
G = Amandin (almonds).
--------------+-------+-------+-------+-------+-------+-------+-------
| A | B | C | D | E | F | G
--------------+-------+-------+-------+-------+-------+-------+-------
Glycine | 0.02 | 0.00 | 0.00 | 0.39 | 3.80 | 0.57 | 0.51
Alanine | 2.00 | 0.43 | 9.79 | 1.15 | 3.60 | 1.92 | 1.40
Valine | 0.21 | 0.13 | 1.88 | 1.36 | 6.20 | 0.26 | 0.16
Leucine | 5.61 | 5.67 | 19.55 | 8.80 | 14.50 | 7.32 | 4.45
Proline | 7.06 | 13.73 | 9.04 | 4.04 | 4.10 | 2.82 | 2.44
Phenylalanine | 2.35 | 5.03 | 6.55 | 2.87 | 3.09 | 3.32 | 2.53
Aspartic acid | 0.58 | ..... | 1.71 | 3.21 | 4.50 | 3.30 | 5.42
Glutamic acid | 42.98 | 43.19 | 26.17 | 18.30 | 18.84 | 12.35 | 23.14
Serine | 0.13 | ? | 1.02 | ? | 0.33 | ? | ?
Cystine | 0.45 | ? | ? | ? | 1.00 | 0.23 | ?
Tyrosine | 1.20 | 1.67 | 3.55 | 2.42 | 2.13 | 3.07 | 1.12
Arginine | 3.16 | 2.16 | 1.55 | 11.06 | 14.17 | 14.44 | 11.85
Histidine | 0.61 | 1.28 | 0.43 | 2.94 | 2.19 | 2.63 | 1.58
Lysine | ..... | ..... | ..... | 3.99 | 1.65 | 1.99 | 0.70
Tryptophane |present|present| absent|present|present|present|present
Ammonia | 5.11 | 4.87 | 3.64 | 2.12 | 2.28 | 1.55 | 3.70
+-------+-------+-------+-------+-------+-------+-------
| 71.46 | 78.16 | 85.27 | 62.65 | 82.38 | 55.77 | 59.00
--------------+-------+-------+-------+-------+-------+-------+-------
At the time when these analyses were made, a method for the quantitative
estimation of tryptophane had not been devised, although one is now
available. The addition of the percentages of tryptophane and of other
amino-acids for which methods of determination are not yet known, would
bring the total, in each case, more nearly up to the full 100 per cent.
These data will serve to show how widely the different plant proteins vary
in the proportions of the different amino-acids which they contain. Animal
proteins have been found to be still more variable in composition.
In the use of the proteins as food for animals, it appears that the
different amino-acids are in some way connected with the different
physiological functions which the proteins have to perform in the animal
body: thus, _tryptophane_ is absolutely essential to the maintenance of
life, but does not promote growth; _lysine_, on the other hand, definitely
promotes growth, so that animals which have been maintained without any
increase in weight for many months immediately begin to grow when furnished
with a diet in which lysine is a constituent; while _arginine_ seems to be
definitely associated with the reproductive function; and _cystine_, with
the growth of hair, feathers, etc. It is not known whether there is any
similar relation of amino-acids to the functions of different proteins in
plant metabolism.
The separation of the individual amino-acids from the mixture which results
from the hydrolysis of any given protein is a long and tedious process and,
at best, yields only moderately satisfactory results. For that reason, it
has recently been almost entirely abandoned in favor of the separation
devised by Van Slyke, which divides the total nitrogenous matter in the
mixture resulting from the hydrolysis of a protein into the following
groups; ammonia N, humin (or melanin) N, cystine N, arginine N, histidine
N, lysine N, amino N of the filtrate, and non-amino N of the filtrate.
These groups can be conveniently and fairly accurately separated out of the
hydrolysis mixture, by means of various precipitating agents, and the
quantity of N in the several precipitates determined by the usual Kjeldahl
method. The actual process for these separations need not be discussed
here, as it is given in detail in all standard text-books dealing with the
methods of biochemical analysis. The distribution of the nitrogen in any
given protein into these various groups is characteristic for that
particular protein, and the process serves both as a means of
identification of individual proteins and a method for tracing their
changes through various vital, or biochemical, transformations.
GENERAL PROPERTIES OF THE PROTEINS
Individual proteins differ slightly in their characteristics, but in
general they are all alike in the following physical and chemical
properties.[5]
=Physical Properties.=--(1) The proteins are all
_colloidal_ in character, that is, they form solutions in water, out of
which they cannot be dialyzed through parchment, or other similar
membranes. (2) All natural proteins, when in colloidal solution, may be
_coagulated_, forming a semi-solid _gel_, which cannot again be rendered
soluble except by decomposition. The most familiar example of this type of
coagulation is that of egg-albumin, when eggs are cooked. This coagulation
may be produced by heat, by the action of certain enzymes, or by the
addition of alcohol to the solution. (3) All solutions of plant proteins
are optically active, rotating the plane of polarized light to the left, in
every case. (4) Proteins are precipitated out of their solutions, without
change in the composition of the protein, by saturating the solution with
various neutral salts of the alkali, or alkaline earth, metals, such as
sodium chloride, ammonium sulfate, magnesium sulfate, etc. This is only
another way of saying that the proteins are insoluble in strong salt
solutions. Separation from solution by the addition of salts is different
from coagulation by heat, etc., as in this case simple dilution of the salt
solution will cause the protein to redissolve, whereas a coagulated protein
cannot be redissolved without some change in its composition.
=Chemical Properties.= (1) Precipitation reactions.--The proteins have both
acid and basic properties (due to the presence in their molecules of both
free NH_{2} groups and free COOH groups). Bodies of this kind are known as
"amphoteric electrolytes," since they yield both positive and negative
ions, if dissociated. The proteins readily form salts, which are generally
insoluble in water, with strong acids. For this reason, they are generally
precipitated out of solution by the addition of the common mineral acids.
They are also precipitated by many of the "alkaloidal reagents," to which
reference has been made in the preceding chapter, namely, phosphotungstic,
phosphomolybdic, tannic, picric, ferrocyanic, and trichloracetic acids, the
double iodide of potassium, mercuric iodide, etc. The precipitates produced
by strong mineral acids are often soluble in excess of the acid, with the
formation of certain so-called "derived proteins," which are probably
products of the partial hydrolysis of the protein.
The proteins are also precipitated out of solution by the addition of small
amounts of salts of various heavy metals, such as the chlorides, sulfates,
and acetates of iron, copper, mercury, lead, etc. This precipitation is
different than that caused by the saturation of the solution with the salts
of the alkali metals, as in this case the metal unites with the protein to
form definite, insoluble salts, which cannot be redissolved except by
treatment with some reagent which removes the metal from its combination
with the protein (hydrogen sulfide is commonly used for this purpose).
(2) Color reactions.--Certain specific groups which are present in most
proteins give definite color reactions with various reagents. It is
apparent that any individual protein will respond to a particular color
reaction, or will not do so, depending upon whether the particular group
which is responsible for the color in question is present in that
particular protein. Color reactions to which most of the common plant
proteins respond are the following ones:
(_a_) _Biuret Reaction._--Solutions of copper sulfate, added to an alkaline
solution of a protein, give a bluish-violet color if the substance contains
two, or more, --CONH-- groups united together through carbon, nitrogen, or
sulfur atoms. Inasmuch as most natural proteins contain several such
groups, the biuret reaction is a very general test for proteins.
(_b_) _Millon's Reaction._--A solution of mercuric nitrate containing some
free nitrous acid (Millon's reagent) produces a precipitate which turns
pink or red, whenever it is added to a solution which contains tyrosine, or
a tyrosine-containing protein.
(_c_) _Xanthoproteic Acid Reaction._--This is the familiar yellow
coloration which is produced whenever nitric acid comes in contact with
animal flesh. It is caused by the action of nitric acid on tyrosine. The
color is intensified by heating, and is changed to orange-red by the
addition of ammonia.
(_d_) _Adamkiewicz's Reaction._--If concentrated sulfuric acid be added to
a solution of a protein to which some acetic acid (or better, glyoxylic
acid) has previously been added, a violet color is produced. This color
will appear as a ring at the juncture of the two liquids, if the sulfuric
acid is poured carefully down the sides of the tube, or throughout the
mixture if it is shaken up. It depends upon the interaction of the
glyoxylic acid (which is generally present as an impurity in acetic acid)
upon the tryptophane group, and is therefore given by all proteins which
contain tryptophane.
(_e_) _Molisch's reaction_ for furfural will be shown by those proteins
which contain a carbohydrate group. In applying this test, the solution to
be tested is first treated with a few drops of an alcoholic solution of
[alpha]-naphthol, and then concentrated sulfuric acid is poured carefully
down the sides of the test-tube. If carbohydrates are present, either free
or as a part of a protein molecule, a red-violet ring forms at the juncture
of the two liquids.
(_f_) _Sulfur Test._--If a drop of a solution of lead acetate be added to a
solution containing a protein, followed by sufficient sodium hydroxide
solution to dissolve the precipitate which forms, and the mixture is heated
to boiling, a black or brown coloration will be produced if the protein
contains cystine, the sulfur-containing amino-acid.
FOOTNOTES:
[5] Since the proteins are essentially _colloidal_ in nature, many of the
terms used in the discussions of their properties, and these properties
themselves, will be better understood after the chapter dealing with the
colloidal condition of matter has been studied. A more logical arrangement
so far as the systematic study of these properties is concerned would be to
take up chapter XV before undertaking the study of the proteins (this order
is actually followed in some texts on Physiological Chemistry). But from
the standpoint of the consideration of the various groups of organic
components of plants, it seems a better arrangement to consider these
groups in sequence, and then to discuss the various physical-chemical
phenomena which govern their activity. However, it is recommended that the
student refer at once to Chapter XV for an explanation of any terms used
here, which may not be familiar to him; and that after the study of Chapter
XV, he return to this chapter dealing with the proteins for an illustrative
study of the applications of the principles presented there.
THE CLASSIFICATION OF THE PROTEINS
Formerly, the classification of proteins was based almost wholly upon their
solubility and coagulation reactions. More recently, since their products
of hydrolysis have been extensively studied, their classification has been
modified, in attempts to make it correspond as closely as possible to their
chemical constitution and physical properties. As knowledge of these
matters progresses, the schemes of classification change. On that account,
no one definite scheme is universally used. For example, the English system
varies considerably from the one commonly used by American biochemists,
which is the one presented below.
The proteins are divided into three main classes, as follows:
(1) Simple proteins, which yield only amino-acids when hydrolyzed.
(2) Conjugated proteins, compounds of proteins with some other non-protein
group.
(3) Derived proteins, decomposition products of simple proteins.
The first two of these classes comprise all the natural proteins; while the
third includes the artificial polypeptides and proteins which have been
modified by reagents.
These major classes are further subdivided into the following sub-classes,
which depend in part upon the solubilities of the individual proteins, and
in part upon the nature of their products of hydrolysis:
1. _The Simple Proteins_
A. Albumins--soluble in water and dilute salt solutions, coagulated
by heat.
B. Globulins--insoluble in water, soluble in dilute salt solutions,
coagulated by heat.
C. Glutelins--insoluble in water or dilute salt solutions, soluble
in dilute acids or alkalies, coagulated by heat.
D. Prolamins--insoluble in water, etc., soluble in 80 per cent
alcohol.
E. Histones--soluble in water, insoluble in ammonia, not coagulated
by heat.
F. Protamines--soluble in water and ammonia, not coagulated by
heat, yielding large proportions of diamino-acids on hydrolysis.
G. Albuminoids--insoluble in water, salt solutions, acids, or
alkalies.
2. _Conjugated Proteins_
A. Chromoproteins--compounds of proteins with pigments.
B. Glucoproteins--compounds of proteins with carbohydrates.
C. Phosphoproteins--proteins of the cytoplasm, containing
phosphoric acid.
D. Nucleoproteins--proteins of the nucleus, containing nucleic
acids.
E. Lecithoproteins--compounds of proteins with phospholipins.
F. Lipoproteins--compounds of proteins with fats, existence in
nature doubtful, artificial forms easily prepared.
3. _Derived Proteins_
A. Primary protein derivatives.
_a._ Proteans--first products of hydrolysis, insoluble in
water.
_b._ Metaproteins--result from further action of acids or
alkalies, soluble in weak acids and alkalies, but
insoluble in dilute salt solutions.
_c._ Coagulated proteins--insoluble forms produced by the
action of heat or alcohol.
B. Secondary protein derivatives.
_a._ Proteoses--products of hydrolysis, soluble in water,
not coagulated by heat, precipitated by saturation of
solution with ammonium sulfate.
_b._ Peptones--products of further hydrolysis soluble in water,
not coagulated by heat, not precipitated by ammonium
sulfate, give biuret reaction.
_c._ Peptides--individual amino-acids, or poly-peptides, may or
may not give biuret reaction.
The plant proteins which have been investigated, thus far, fall into these
groups as follows:
1A. _Albumins_
Leucosin, found in the seeds of wheat, rye, and barley.
Legumelin, " " pea, horse-bean, vetch, soy-bean,
lentil, cowpea, adzuki-bean.
Phaselin, " " kidney-bean.
Ricin, " " castor-bean.
1B. _Globulins_
Legumin, found in the seeds of pea, horse-bean, lentil and vetch.
Vignin, " " cowpea.
Glycinin, " " soy-bean.
Phaseolin, " " beans (_Phaseolus spp._)
Conglutin, " " lupines.
Vicilin, " " pea, horse-bean, lentil.
Corylin, " " hazel nut.
Amandin, " nuts of almond and peach.
Juglansin, " seeds of walnut and butternut.
Excelsin, " " Brazil nut.
Edestin, " hemp seed.
Avenalin, " oats.
Maysin, " corn.
Castanin, " the seeds of European chestnut.
Tuberin, " potato tubers.
And, crystalline globulins found in the seeds of flax, squash, castor-bean,
sesame, cotton, sunflower, radish, rape, mustard, and in cocoanuts,
candlenuts, and peanuts.
1C. _Glutelins_
Glutenin, found in the seeds of wheat.
Oryzenin, " " rice.
1D. _Prolamins_
Gliadin, found in the seeds of rye, wheat, with glutenin forms
"gluten."
Hordein, " " barley.
Zein, " " corn.
1E-1G. _Histones, Protamines and Albuminoids._--So far as is now known, no
representatives of these classes are found in plants.
2. Conjugated Proteins.--There is no conclusive evidence of the existence
in plants of any of the conjugated proteins, other than the nucleoproteins
and the chromoproteins, the composition and properties of which have been
discussed in previous chapters. The nucleoproteins undoubtedly occur in the
embryos of many, if not all, seeds.
3. Derived Proteins.--Representatives of the various types of derived
proteins are undoubtedly found as temporary intermediate products in
plants, both as products of hydrolysis produced during the germination of
seeds and as intermediate forms in the synthesis of proteins. So far as is
known, however, they do not occur as permanent forms in any plant tissues.
They have been prepared in large numbers and quantities, by the hydrolysis
of the natural proteins and the artificial synthesis of polypeptides.
In the present state of our knowledge concerning the functioning of the
proteins, no significance in the physiology of plant life, or metabolism,
is to be attached to the particular type of protein material which it
contains, at least so far as the simple proteins of the cytoplasm are
concerned.
DIFFERENCES BETWEEN PLANT AND ANIMAL PROTEINS
A much larger variety of protein materials is found in animal tissues than
in plants. This is undoubtedly because different animal organs perform so
much more varied physiological functions than do those of plants. Three
groups of simple proteins, the histones, the protamines, and the
albuminoids, which are quite common in animal tissues, are entirely unknown
in plants. Further, conjugated proteins of greater complexity and more
varied structure are found in animal tissues, especially in the brain,
nerve-cells, etc., than in plants.
Plant proteins, in general, usually contain larger proportions of proline
and of glutamic acid than are found in animal proteins; also more arginine
than is found in any of the animal proteins except the protamines, which
contain as high as 85 per cent of this amino-acid.
Of the twenty-five plant proteins which have thus far been hydrolyzed and
studied from this standpoint, all contained leucine, proline,
phenylalanine, aspartic acid, glutamic acid, tyrosine, histidine, and
arginine; two gave no glycine; two others, no alanine; four contained no
lysine; and one, no tryptophane. Zein, the principal protein of corn
contains no glycine, lysine, or tryptophane. It is not sufficient to
support animal life and promote growth, if used as an exclusive source for
protein for food.
THE EXTRACTION OF PROTEINS FROM PLANT TISSUES
Since proteins are indiffusible, it is essential that the cell-walls of the
tissue shall be thoroughly ruptured as the first step in any process for
the extraction of these compounds from plant tissues. This is usually
accomplished by grinding the material as finely as possible, preferably
with the addition of sharp quartz sand, or broken glass, to aid in the
tearing of the cell-wall material.
The solvent to be used in extracting the proteins from this finely ground
material depends upon the nature and solubility of the proteins which are
present, and also upon whether it is desired to separate the proteins which
may be present in the plant, during the process of the extraction. A glance
at the scheme of classification of the proteins will show the following
solubilities which serve as a guide to the procedure to be followed: (_a_)
proteoses, albumins, and some globulins may be extracted with water; (_b_)
globulins and most of the water-soluble proteins may be extracted by using
a 10 per cent solution of common salt; (_c_) prolamines are extracted by
70-90 per cent alcohol; glutelins and prolamins dissolve in dilute acids or
dilute alkali.
A common procedure is to extract groups (_a_) and (_b_), using a 10 per
cent salt solution as the solvent, and then to separate the albumins,
globulins, etc., from this solution by suitable precipitants; then to treat
the material with 80 per cent alcohol, to extract the prolamines; and
finally with dilute alkali, to extract the glutelins. The dissolved
proteins in each extract can be subsequently purified by dialysis,
precipitation, etc. The insoluble proteins can be studied only after
removing the other materials associated with them in the tissue, by
suitable mechanical or chemical means.
THE SYNTHESIS OF PROTEINS IN PLANTS
The synthesis of proteins in plants is not a process of photosynthesis, as
it can take place in the dark and in the absence of chlorophyll, or any
other energy-absorbing pigment. However, protein-formation normally takes
place in conjunction with carbohydrate-formation. The carbon, hydrogen,
and oxygen necessary for protein synthesis are undoubtedly obtained from
carbohydrates. The nitrogen and sulfur come from the salts absorbed from
the soil through the roots and brought to the active cells in the sap.
Atmospheric nitrogen cannot be used by plants for this purpose, except in
the case of certain bacteria and other low plants, notably the bacteria
which live in symbiosis with the legumes in the nodules on the roots of the
host plants. In general, the sulfur must come in the form of sulfates and
the nitrogen in the form of nitrates; although many plants can make use of
ammonia for protein-formation. Presumably, the nitrate nitrogen must be
reduced in the plant to nitrites, and then to ammonia form, in order to
enter the amino-arrangement required for the greater proportion of the
protein nitrogen.
The mechanism by which ammonia nitrogen becomes amino-acids in the plant is
not understood. Artificial syntheses of amino-acids, by the action of
ammonia upon glyoxylic acid and sorbic acid, both of which occur in plants
and may be obtained by the oxidation of simple sugars, have been
accomplished, and it seems probable that similar reactions in the plant
protoplasm may give rise to the various amino-acids which unite together to
form proteins. Nothing is known, however, of the process by which the more
complicated closed-ring amino-acid compounds, such as proline, histidine,
or tryptophane, are synthetized.
The condensation of amino-acids into proteins, or the reverse
decomposition, is very readily accomplished in all living protoplasm, under
the influence of special protein-attacking enzymes, which are almost
universally present in the cytoplasm. These reactions in connection with
the proteins are similar to the easy transformation of sugars to starches,
and _vice versa_, under the action of the corresponding
carbohydrate-attacking enzymes.
PHYSIOLOGICAL USES OF PROTEINS
There can be no doubt that the all-important rôle of proteins, in either
plant or animal tissue, is to furnish the colloidal protoplasmic material
in which the vital phenomena take place. Their occurrence in seeds, and
other storage organs, is, of course, in order to provide the
protoplasm-forming material for the young seedling plant.
They are, moreover, the source for the material which goes into some of the
secretion groups of organic compounds; as they are easily broken down by
various agents of decomposition into nitrogen-free alcohols, aldehydes, and
acids, which produce the essential oils, pigments, etc.
Much, if not all, of their physiological activity is due to their colloidal
nature, the importance and effects of which will be more apparent after the
chapters dealing with the colloidal condition of matter and with the
physical chemistry of protoplasm have been studied.
REFERENCES
ABDERHALDEN, E.--"Neuere Ergebnisse auf dem Gebiete der Speziellen
Eiweisschemie," 128 pages, Jena, 1909.
FISCHER, E.--"Untersuchungen über Aminosäuren, Polypeptide, und Proteine,
1899-1906," 770 pages, Berlin, 1906.
MANN, G.--"Chemistry of the Proteids," 606 pages, London, 1906.
OSBORNE, T. B.--"The Vegetable Proteins," 138 pages, _Monographs_ on
Biochemistry, London, 1909.
PLIMMER, R. H. A.--"The Chemical Constitution of the Proteins, Part I,
Analysis," 188 pages; and "Part II, Synthesis, etc." 107 pages,
_Monographs_ on Biochemistry, London, 1917. (3d ed.).
ROBERTSON, T. B.--"The Physical Chemistry of the Proteins," 477 pages, New
York, 1918.
SCHRYBER, S. B.--"The General Characters of the Proteins," 86 pages,
_Monographs_ on Biochemistry, London, 1909.
UNDERHILL, F. P.--"The Physiology of the Amino-acids," 169 pages, 13 figs.
1 plate. Yale University Press, 1915.
CHAPTER XIV
ENZYMES AND THEIR ACTION
The characteristic difference between the reactions of inorganic compounds
and those of organic substances lies in the rapidity, or velocity, of the
chemical changes involved. Speaking generally chemical reactions take place
between substances which are in solution, so that they may come into
sufficiently intimate contact that chemical action between them can take
place. There are, of course, occasional examples of reactions between dry
solids, such as the explosion of gunpowder, etc., but the general rule is
that reacting materials must be in either colloidal or true solutions.
Inorganic materials, when dissolved in water, usually ionize very readily.
That is, they are not only disintegrated into individual _molecules_, but a
considerable proportion of these molecules separate into their constituent
_ions_. When solutions containing ionized compounds are brought together,
conditions for chemical interaction are ideal, and the reaction proceeds
with such tremendous rapidity as to be completed almost instantaneously, in
most cases.
Organic compounds, on the other hand, ionize only very slowly, if at all.
Hence, reactions between organic compounds, even when they are in solution,
proceed very slowly unless carried on at high temperatures, under increased
pressure, or under the influence of some catalytic agent. Even under the
stimulation of these reaction-accelerating agencies, most chemical changes
in organic compounds when carried on in the laboratory, require several
hours or even days and sometimes weeks, for their completion. But when
similar reactions take place in living organisms, they proceed with
velocities which resemble those of inorganic compounds in the laboratory.
This difference between the velocity of organic reactions when carried on
under artificial conditions in the laboratory (often spoken of as "_in
vitro_") as compared with that of the same reactions when they take place
in a living organism ("_in vivo_"), is due to the universal presence in
the living protoplasm of certain organic catalysts, known as _enzymes_.
ENZYMES AS CATALYSTS
The phenomenon known as "catalysis" is of common occurrence in both
inorganic and organic chemistry. The effect of a small amount of manganese
dioxide in aiding in the liberation of oxygen from potassium chlorate is an
example which is familiar to all students of elementary chemistry.
Similarly, spongy platinum accelerates the oxidation of sulfur dioxide to
sulfur trioxide, in the commercial manufacture of sulfuric acid. Again, the
hydrolysis of sucrose into fructose and glucose proceeds very slowly in the
presence of water alone, but if a little hydrochloric acid or sulfuric acid
be added to the solution, the velocity of the hydrolysis is enormously
accelerated. Many other examples of the accelerating effect of various
chemicals upon reactions into which they do not themselves enter, might be
cited.
The essential features of all such catalytic actions are: (1) the velocity
of the reaction is greatly altered, usually accelerated; (2) the catalytic
agent does not appear as one of the initial substances, or end-products, of
the reaction, and is not itself altered by the chemical change which is
taking place; (3) the accelerating effect is directly proportional to the
amount of the catalyst which is present; (4) relatively small amounts of
the catalyst produce very large results in the reacting mixture; and (5)
the catalysts cannot themselves initiate reactions, but only influence the
velocity of reactions which would otherwise take place at a different rate
(usually much more slowly) in the absence of any catalytic agent.
Enzymes conform to all of these properties of catalysts, and are commonly
defined as the "catalysts of living matter." They are almost universally
present in living organs of every kind, and perform exceedingly important
functions, both in the building-up of synthetic materials and in the
rendering soluble of the food of both plants and animals, so that it can be
translocated from place to place through the tissues of the organism.
Enzymes differ from inorganic catalysts in being destroyed by heat, in not
always carrying the reaction to the same stage as does the inorganic
catalyst which may accelerate the same reaction, and in producing
different changes in the same substance by different enzymes.
The name "enzyme" comes from Greek words meaning "in yeast," as the nature
and effect of the enzyme involved in the alcoholic fermentation of sugars
by yeast were those which were first recognized and understood. It was at
first thought, by Pasteur and his students, that fermentation is the direct
result of the life activities of the yeast plant. Later, it was found that
water extracts from sprouted barley, from almond seeds, and from the
stomach, pancreas, etc., were able to bring about the decomposition of
starch, of amygdalin, and of proteins, respectively, in a way which seemed
to be quite comparable to the fermentative action of yeasts. Hence, it was
thought that there were two varieties of active agents of this kind, one
composed of living cells and the other non-living chemical compounds, and
these were called the "organized ferments" and the "unorganized ferments,"
respectively. However, in 1897, Büchner found that by grinding yeast cells
with sharp sand until they were completely disintegrated and then
submitting the mass to hydraulic pressure, he could obtain a clear liquid,
entirely free from living cells, which was just as active in producing
fermentation as was the yeast itself. This discovery paved the way for a
long series of investigations, which have conclusively demonstrated that
there is no distinction between "organized" and "unorganized" ferments,
that all living organisms perform their characteristic functions by means
of the enzymes which they contain, and that these enzymes can bring about
their characteristic catalytic effects outside the cell, or tissue which
elaborates them, just as well as within it, provided only that the
conditions of temperature, acidity or alkalinity of the medium, etc., are
suitable for the particular enzyme action which is under consideration.
GENERAL PROPERTIES OF ENZYMES
Since enzymes are catalysts, it is plain that an accurate description of
their activity should, in each case, refer to the influence which they
exert upon some definite reaction velocity. But since the phrases necessary
to describe such an effect are cumbersome and inconvenient, and since most
of the reactions which are accelerated by the catalytic action of enzymes
are either simple hydrolyses, changes in oxygen content, or other simple
decompositions or condensations, which will otherwise proceed so slowly as
to be practically negligible, it is customary to speak of the enzyme as
"acting upon" the material in question. It should be understood, however,
that this is a misstatement, as the enzyme cannot actually initiate a
reaction, or "act upon" any substance; it only acts as a catalyzer to
accelerate the action of water, oxygen, etc., upon the material in
question.
Generally speaking, most enzymes are colloidal in form and, hence, do not
diffuse through membranes such as the cell-walls. Some of them perform
their characteristic functions only within the cell, or organ, which
elaborates them, and can be obtained outside these tissues for purposes of
study only by first rupturing the cell-wall or other membrane with which
they are surrounded. Such enzymes are known as "intracellular." Others are
regularly secreted by glands which discharge them onto other organs, as the
stomach or intestines of animals, where they perform their useful
functions; or, as in the case of germinating seeds, they move to other
parts of the organ, and can be extracted from the tissue by simple
treatment with water. These are known as the "extracellular" enzymes.
Enzymes are specific in their action. Any given enzyme affects only a
single reaction; or at most acts only upon a single group of compounds
which have similar molecular configuration. Usually it is only a single
compound whose decomposition is accelerated by the action of a particular
enzyme; but there are a few enzymes, such as _maltase_ (which acts on all
[alpha]-glucosides) and emulsin (which acts on all [beta]-glucosides) which
act catalytically upon groups of considerable numbers of similar compounds.
Enzymes, like all other catalysts, act more energetically at increased
temperatures; but for each particular enzyme there is an "optimum
temperature," (usually between 40° and 65°) above which the destructive
effect of the temperature upon the enzyme itself more than offsets the
accelerating influence of the increased temperature. At still higher
temperatures (usually 80° to 100°) the enzymes are "killed," i.e., rendered
permanently inactive. All enzymes are "killed" by boiling the solutions in
which they are contained. Dry preparations of enzyme material can withstand
somewhat higher temperatures, for somewhat longer periods of time, than can
the same enzyme in moist condition or in solution. When an enzyme has once
been inactivated by heating, or "killed," it can never be restored to
activity again.
Enzymes are extremely sensitive to acids, bases, or salts, their activity
being often enormously enhanced or, in other cases, entirely inhibited, by
the presence in the reacting medium of very small amounts of free acids, or
bases, or even of certain neutral salts. For example, pepsin, the enzyme of
the stomach will act only in the presence of a slightly acid medium and is
wholly inactive in a mixture which contains even the slightest amount of
free alkaline material; while trypsin, the similar enzyme of the intestine,
acts only under alkaline conditions. Practically all enzymes are rendered
inactive, but not destroyed, by the presence of either acid or alkali in
excess of N/10 strength. Many will act only in the presence of small
quantities of certain specific neutral salts; while, on the other hand,
other salts are powerful inhibitors of enzyme action. Enzymes often differ
from the protoplasm which secretes them in their response to antiseptics,
such as toluene, xylene, etc., which inhibit the activity or growth of the
cell, but have no effect upon the activity of the enzymes which it
contains.
THE CHEMICAL NATURE OF ENZYMES
Nothing is known with certainty concerning the chemical nature of enzymes.
Being colloidal in nature, they adsorb carbohydrates, proteins, fats, etc.,
so that active enzyme preparations often respond to the characteristic
tests for these groups of substances; and many investigators have reported
what has, at first, seemed to be conclusive evidence that some particular
enzyme which they have studied is either a carbohydrate, a protein, or some
other type of organic compound. Later investigations have always shown,
however, that if the preparation in question be submitted to the digestive
action of the enzymes which hydrolyze the particular type of substances to
which it is supposed to belong, the material will lose its characteristic
protein, or carbohydrate, etc., properties, without losing its specific
activity, thus clearly indicating that the substance which responds to the
characteristic tests for some well-known type of organic compounds is
present as an impurity and is not the enzyme itself.
The present state of knowledge concerning the nature of enzymes seems to
indicate that, like the inorganic catalysts, they may vary widely in
chemical composition; and that their tremendous catalytic effects are due,
in part at least, to their colloidal nature. This will be better understood
and appreciated after the phenomena associated with the colloidal condition
have been considered (see the following chapter).
NOMENCLATURE AND CLASSIFICATION
Since nothing is known of the chemical composition of enzymes, they can
only be studied by considering the effects which they produce. This is
reflected in the systems which have been adopted for their nomenclature and
classification.
As they were first supposed to be proteins, the earlier representatives of
the group were given characteristic names ending with the suffix _in_,
similar to that of the proteins. Since this idea has been found to be
incorrect, however, a system of nomenclature has been adopted which assigns
to each enzyme the name of the material upon which it acts, followed by the
suffix _ase_. Thus, cellulase is the enzyme which accelerates the
hydrolysis of cellulose; glucase, that acting upon glucose; amylase, that
acting upon starch (_amylum_), etc.
The substance upon which the enzyme acts (or, strictly speaking, the
substance whose hydrolysis, oxidation, or other chemical change, is
catalytically affected by the enzyme) is called the _substrate_.
Most enzymes are catalysts for hydrolysis reactions and are, hence, classed
as _hydrolytic_ in their action, and may be spoken of as "hydrolases."
Those which accelerate oxidation are called "oxidases"; while those that
stimulate reduction reactions are "reductases"; those that aid in the
splitting off of ammonia, or amino-acid groups, are "deaminases"; and those
that aid in the splitting off of CO_{2} from COOH groups are
"carboxylases," etc.
The hydrolytic enzymes are further subdivided into the sucroclastic
(sugar-splitting), or sucrases; the lipoclastic (fat-splitting), or
lipases; the esterases (ester-splitting); proteoclastic
(protein-splitting), or proteases; etc.
OCCURRENCE AND PREPARATION FOR STUDY
Enzymes are present in all living matter. In animal tissues, they occur in
the largest amounts in those glands or organs where active vital processes
take place, as in the brain, the digestive tract, blood, etc. In plants,
they may be found in all living cells, and are especially abundant in the
seeds, where they serve to render soluble and available to the young plant
the stored food materials. The enzymes of moulds, and other parasitic
plants, are usually extracellular in type, being secreted for the purpose
of making the material of the host plant available to the parasite.
Extracellular enzymes are also developed in seeds during germination, in
order that the stored food material of the endosperm may be rendered
soluble and translocated into the tissues of the growing seedling. But most
other plant enzymes are intracellular in type. Hence, in all preparations
of plant enzymes for study, or for commercial use, the first step in the
process is, necessarily, a thorough rupturing of the cell-walls of the
plant material.
The rupturing of the cells may be accomplished in a variety of ways, as
follows: (1) mechanical disintegration, as by grinding in a mortar with
sharp sand; (2) freezing the material, by treatment with liquid air, then
grinding; (3) killing the cells by drying, by treatment with alcohol or
acetone, then grinding the mass in a paint mill with toluene; (4) killing
the cells by chemicals (sulfuric acid, 0.5 to 1.0 per cent, or other
suitable agents) followed by extraction with water; (5) autolysis, or
self-digestion, in which the cells are mixed with toluene or some other
antiseptic which kills the cells without injuring the enzymes, then the
material is minced or ground up and suspended in water containing the
antiseptic, until the enzymes dissolve the cell-walls and so escape into
the liquid--this process being especially adapted to the preparation of
active extracts from yeasts, which contain the necessary cell-wall
dissolving enzymes to facilitate autolysis.
Enzymes may be separated out of the aqueous extract obtained from cells
ruptured by any of the above methods, by precipitation with alcohol,
acetone, or ether, in which they are insoluble; but if this is done, the
precipitate must be at once filtered off and rapidly washed and dried, as
prolonged contact with these precipitating agents greatly diminishes the
activity of most enzymes. Or, they may be adsorbed out of solution on
gelatinous, or colloidal, materials, like aluminium hydroxide, or various
hydrated clays. If the dry preparations obtained in any of these ways are
contaminated by carbohydrates, proteins, etc., these may be removed by
treatment with suitable digesting enzymes obtained from the saliva,
gastric, and pancreatic juices, and the digested impurities washed out with
60 to 80 per cent alcohol, leaving the enzyme preparation in a purified but
still active form.
In any study of the "strength," or possible catalytic effects, of an enzyme
preparation, it is necessary, first, to determine what particular reaction
it affects, by qualitative tests with various substrate materials, such as
starch, sugars, glucosides, proteins, etc., and then to determine
quantitatively its accelerating effect upon the reaction in question. The
latter may be done by measuring either the _time_ required to carry a unit
quantity of the substrate material through any determined stage of chemical
change, or the _quantity_ of the substrate which is changed in a unit
period of time. It would not be profitable to go into a detailed discussion
here of the methods of making these quantitative measurements of enzyme
activity. Such discussions must necessarily be left to special treatises on
methods of study of enzyme action. It may be said, however, that generally
both the qualitative tests for, and the quantitative measurements of, the
accelerating influence of enzymes depend upon the observation of some
change in the physical properties of the substrate material, such as the
optical activity, electrical conductivity, or viscosity, of its solution.
In some cases, it is convenient to make an actual quantitative
determination of the amount of end-products produced in a given time, as in
the inversion of cane sugar, the hydrolysis of maltose, etc., but such
determinations necessarily involve the removal of some of the reaction
mixture for the purposes of the determinations, and are not, therefore,
suitable for the study of the progressive development of the reaction which
is being studied.
Enzymes are found in all parts of the animal organism and those which are
active in the digestion of food, the metabolism of digested material, the
coagulation of blood, etc., have been extensively studied. A discussion of
these animal enzymes would be out of place in such a text as this, however,
and the following list includes only enzymes which are known to occur in
plant tissues. These well-known enzymes will serve as examples of the
several general types which have thus far been isolated and studied.
------------------- -+-----------+-------------+-------------+-----------
Class and Type. | Enzyme. | Substrate. |End-products.| Found in.
---------------------+-----------+-------------+-------------+-----------
I. Hydrolases | | | |
(_a_) Esterases |Lipase |Fats |Glycerol and |Oily seeds
| | |fatty acids |
| | | |
(_b_) Carbohydrases |Sucrase or |Sucrose |Glucose and |Yeasts
|invertase | |fructose |
| | | |
|Maltase |Maltose and |Glucose, etc.|Barley malt
| |all [alpha]- | |
| |glucosides | |
| | | |
|Dextrinase |Dextrin |Maltose |Malt
| | | |
|Inulase |Inulin |Fructose |Artichokes,
| | | |etc.
| | | |
|Amylase or |Starch |Maltose |Malt, etc.
|diastase | | |
| | | |
|Cellulase |Cellulose |Maltose |Bacteria
| | | |
|Pectinase |Pectose |Arabinose |Fruits
| | | |
|Cytase |Hemi- |Mono- |Nuts,
| |celluloses |saccharides |seeds, etc.
| | | |
(_c_) Glucosidases |Emulsin |Amygdalin |Glucose, |Almond
| |and all |etc. |kernels,
| |[beta]- | |etc.
| |glucosides | |
| | | |
|Maltase |All [alpha]- |Glucose, |Barley malt
| |glucosides |etc. |
| | | |
|Myrosin |Sulfur- |Glucose, |Mustard
| |containing |etc. |seeds
| |glucosides | |
| | | |
|Rhamnase |Xanthorhamnin|Rhamnose, |_Rhamnus
| | |etc. |spp._
| | | |
|Phytase |Phytin |Inosite and |Bran coats
| | |H_{3}PO_{4} |of seeds
| | | |
(_d_) Proteases |Erepsin |Proteins |Amino-acids |Many plants
| | | |
|Papain |Proteins |Amino-acids |Papaws
| | | |
|Bromelin |Proteins |Amino-acids |Many plants
| | | |
|Nuclease |Nucleo- |Proteins and |Many plants
| |proteins |nucleic acid |
| | | |
II. Oxidases | | | |
(_a_) Catalases | ........ |Hydrogen |Water and |Nearly
| |peroxide |oxygen |all plants
| | | |
(_b_) Peroxidases | ........ |Organic |"Active" |Nearly
| |peroxides |oxygen |all plants
| | | |
(_c_) Oxidases | ........ |Chromogens |Pigments |Many plants
| | | |
| |Alcohols and |Acids |Many plants
| |phenols | |
(_d_) Reductases | ........ | ......... | ......... |Many plants
| | | |
III. Deaminases |Urease |Urea |Ammonia and |
| | |CO_{2} |
| | | |
|Guanase |Guanine |Xanthine |
| | | |
|Adenase |Adenine |Hypoxanthine |
| | | |
IV. Carboxylases | | | |
(_a_) | ........ |Keto-acids |Aldehydes |
| | |and CO_{2} |
| | | |
(_b_) | ........ |Amino-acids |Amines and |
| | |CO_{2} |
| | | |
V. Coagulation |Pectase |Coagulates | .......... |Fruits
enzymes | |pectic | |
| |bodies | |
| | | |
VI. Fermentation |Zymase |Glucose, etc.|Alcohol and |Yeasts
enzymes | | |CO_{2} |
| | | |
|Lactic acid|Fatty acids |Lactic acid |Bacteria
|ferment | | |
| | | |
|Butyric |Fatty acids |Butyric acid |Bacteria
|acid | | |
|ferment | | |
| | | |
---------------------+-----------+-------------+-------------+----------
The above list includes only the more common and best-known plant enzymes.
It seems reasonable to suppose that for every individual type of organic
compound which may occur in general plant groups, or even in single
species, there is a corresponding enzyme available to affect its
physiological alterations. Indeed, new preparations of active enzymes from
special types of plants and new evidences of the existence of enzymes in
various plant organisms are continuously being reported.
A few of the most common specific representatives of individual groups of
enzymes may be briefly described, as follows:
=Amylase= (or =diastase=, as it was first named and is still commonly
called) is probably the most widely distributed enzyme of plants. It is
found in practically all bacteria and fungi; in practically all seeds (it
has been found in active form in seeds which were known to be over fifty
years old); in all roots and tubers; and in practically all leaves, where
it is located in the stroma of the chloroplasts.
It appears to exist in two modifications, known, respectively, as (_a_)
translocation diastase and (_b_) diastase of secretion. The first form is
found in the cells of ungerminated seeds, in leaves, shoots, etc. It
remains in the cells where reserve starch is stored and aids in the
transformation of starch into soluble materials for translocation from cell
to cell. It is active at a lower temperature than the second form, its
optimum temperature being 45° to 50°. The second form is secreted by the
scutellum, and perhaps by the aleurone cells, of germinating seeds, being
produced by special glandular tissue. It aids in the hydrolysis of the
starch for the use of the growing embryo. Its optimum temperature is 50° to
55°.
The activity of amylase is accelerated by the presence of small quantities
of neutral salts, especially by sodium chloride and disodium phosphate. It
acts best in neutral solutions, its activity being inhibited, although the
enzyme itself is not destroyed, by the presence of more than minute traces
of free mineral acid or alkali.
=Sucrase= (or invertase) is present in almost all species of yeasts, where
it serves to convert unfermentable sucrose into glucose and fructose, which
are readily fermentable. Invertase is also present in moulds and other
microorganisms; and in the buds, leaves, flowers, and rootlets of those
higher order plants which store their carbohydrate reserves in the form of
sucrose. It appears that sucrose, while easily soluble, is not readily
translocated, or utilized, by plants until after it has been hydrolyzed
into its constituent hexoses.
The optimum temperature for invertase is 50° to 54°; it is killed if
heated, in the moist condition, to 70°. Its activity is increased by the
presence of small amounts of free acids; but is inhibited by free alkalies.
=Zymase= is the active alcoholic fermentation enzyme of yeasts. It
accelerates the well-known reaction for the conversion of hexose sugars
into alcohol and carbon dioxide, namely,
C_{6}H_{12}O_{6} = 2C_{2}H_{5}OH + 2CO_{2}.
Because of its scientific interest and industrial importance in the
fermentation industries, its action has been extensively studied. It acts
only in the presence of soluble phosphates and of a coenzyme (see below)
which is dialyzable and not destroyed, which is probably an organic ester
of phosphoric acid. The significance of the molecular configuration of the
hexose sugars in their susceptibility to action by zymase has already been
discussed in detail (see page 56).
The optimum temperature for zymase action is 28° to 30°. The enzyme is
killed by heating to 45° to 50° in solution, or to 85° if in dry
preparation.
=Proteases= of the erepsin type, i.e., those which break proteins down to
amino-acids instead of only to the proteose or peptone stage, as is
characteristic of the enzymes of the trypsin type, are widely distributed
in plants. Except in the case of the two which occur in large amounts in
certain special fruits (papain in papaws, and bromelin in pineapples), they
are very difficult to prepare in pure form for study. In general, all
proteolytic actions, even when accelerated by active enzymes, proceed much
more slowly than do the hydrolyses of carbohydrates or fats. It seems that
metabolic changes of the complex protein molecules are much more difficult
to bring about and take place much more slowly than do those of the
energy-producing types of compounds.
The presence of proteolytic enzymes in most vegetative cells, and in seeds,
may be demonstrated, however, by studying the action of extracts of these
tissues upon soluble proteins. The best-known example of this type of
enzymes is the protease of yeast; but similar ones may be found in
germinating seeds. These vegetable proteases are usually most active in
neutral or only faintly alkaline solutions, and their activity is nearly
always inhibited by even traces of free acids.
Most laboratory studies of proteolytic enzymes are carried on with
preparations of the powerful members of this class of enzymes which are
found in the digestive tract of animals, namely, the pepsin of the gastric
juice, which acts in the acid medium, in the stomach, and the trypsin of
the pancreatic juice, which acts in the alkaline medium of the intestinal
tract. But even these powerful proteases require several hours for the
transformation of an amount of soluble albumin into its amino-acid
constituents which is equivalent to the amount of starch which is
hydrolyzed to maltose by diastase in a very few minutes.
Enzymes which govern oxidative changes, known respectively, as _catalases_
and _oxidases_, are almost universally present in plants. Catalase
decomposes peroxides, with the liberation of free oxygen. It is, therefore,
necessary to the final step in the process of photosynthesis, as elucidated
by Usher and Priestley (see page 26), and serves to prevent the destructive
action of hydrogen peroxide upon chlorophyll. The almost universal presence
of oxidases in plant tissues has been repeatedly demonstrated. They are
present in especially large amounts in tissues which are being acted upon
by parasitic fungi or are combating unfavorable conditions of growth. The
oxidases, in such cases, seem to be the agents by which the plant is able
to stimulate its metabolic activities to overcome the unfavorable
environment for its normal development.
In vegetables and fruits, the common browning, or blackening, of the
tissues when cut surfaces are exposed to the air has been demonstrated to
be due to the catalytic oxidation of the tannins or of certain amino-acids,
especially tyrosine, under the influence of the oxidases which are present
in the tissues. In fact, most pigmentation phenomena are due to changes in
the oxygen content of the chromogens of the cells of the plant, under the
influence of the oxidases which are present in the protoplasm of the cells
in question. Hence, the oxidases may be said to be the controlling agencies
for both the energy-absorbing activities and for respiration in plants.
THE NATURE OF ENZYME ACTION
The mechanism by which an enzyme accomplishes its catalytic effects has
been the object of extensive studies during recent years, especially since
the discovery by Büchner that enzymes could be isolated in solutions
entirely free from the disturbing influence of growing cells. Several
theories concerning the mode of this catalytic action have been advanced.
The earliest and simplest of these was that the enzyme simply creates an
environment favorable for the particular chemical reaction to take place,
as by exposing large surfaces of the substance in question to the action of
the hydrolytic, or other effective, agent, by means of surface adsorption
of the substrate material on the colloidal enzyme.
However, more recent investigations clearly indicate that there is an
actual combination between the substrate material and the enzyme, which
combination then breaks down with a resultant change in the substrate
material and a freeing of the enzyme for repeated recombination with
additional substrate, with the net result that the chemical change in the
substrate material is enormously accelerated. That such a combination
between substrate and enzyme actually exists has been demonstrated in two
different ways: (_a_) experimentally, by mixing together solutions of an
enzyme and of its substrate, each of which is filterable through paper or
through a porous clay filter, with the result that the active material in
the combined solutions will not pass through these same filters; and (_b_)
mathematically, by a study of the curves representing the reaction
velocities of typical reactions which are proceeding under the influence of
an enzyme, which show that so long as there is a large excess of substrate
material present, the accelerating influence of the catalyst is uniform
over given successive periods of time, but that when the quantity of
substrate material becomes smaller than that which permits the maximum
combining power of the enzyme to be exercised, the reaction velocity
immediately slows up.
Again, the fact that the specificity of the action of an enzyme, i.e., the
limitation of the action of that enzyme to a specific single compound or
group of similar compounds, is definitely related to the molecular
configuration of the molecule of the substrate, as has been found to be
true in all those cases where the molecular configuration of the substrate
material has been established (see pages 56 to 58), is an added indication
that there is some kind of a union between the enzyme and the substrate as
a first step in the catalytic process.
As to the nature of this supposed combination of substrate and enzyme, two
theories are held. The first is that this union is in the form of an actual
molecular combination, or chemical compound, and the other is that it is a
purely physical, or colloidal complex. The latter view has by far the
greater weight of theoretical and experimental evidence in its support. The
relation of electrolytes to the catalytic effect of enzymes, the appearance
of the reacting masses under the ultramicroscope, and the effect of heat
upon the reacting mixtures, all point to the conclusion that the phenomenon
is colloidal rather than molecular in character. This view also makes the
remarkable catalytic effects which take place in living protoplasm, which
undoubtedly exists in the colloidal condition, much more easily understood.
This phase of the matter will be much more apparent after the chapter
dealing with the physical chemistry of the protoplasm has been studied.
A further indication that the mechanism of enzyme activity is colloidal in
character lies in the fact that, so far as is known, all reactions which
are catalyzed by specific enzymes are reversible and the same enzyme will
accelerate the velocity of the reaction in either direction, the direction
in which the reaction goes being determined by the conditions surrounding
the reacting material at the time. It was formerly supposed that enzymes
catalyze only decomposition reactions and that the synthetic reactions of
living tissues are produced by means of some other force or agency. This
view supported the idea of a chemical union of the enzyme with the
substrate which, when it breaks down, breaks the molecule of the substrate
material into some simpler form, or forms. But it is now known that the
reaction which is influenced by the enzyme will be catalyzed in either
direction by the specific enzyme which "fits" the particular substrate
material at every point of its molecular configuration, as the glove fits
the hand. The contrast between this fitting of the enzyme to the entire
configuration of the molecule, and the union at a single point or group
which is characteristic of chemical linkages, is apparent. As examples of
the synthetic action of the same enzyme which, under other conditions,
accelerates the decomposition of the same material, there may be cited the
demonstrated synthesis of isomaltose from glucose by maltase; the
production of ethyl butyrate from alcohol and butyric acid; and the
synthetic production of artificial fats, by the aid of the pancreatic
lipase; and the apparent synthesis of a protein from the same amino-acids
which may be obtained from it by hydrolysis under the influence of the same
protease, but under different environmental conditions.
ACTIVATORS AND INHIBITORS
The activity of enzymes is strongly influenced by the presence in the
solution of other bodies, usually, although not always, electrolytes. This
is probably due, in most cases at least, to the action of the electrolyte
upon the colloidal condition of the enzyme. All enzymes do not respond
alike to the action of the same electrolyte, however. The activity of
certain enzymes is enormously increased by the presence of a small amount
of acid; while the action of another may be absolutely inhibited by the
same acid in the same concentration. Thus, the activity of the amylase
found in the endosperm of many seeds is instantly stopped by adding to the
solution enough sulfuric acid to make it two-hundredth normal in strength;
while the same concentration of acid actually accelerates the activity of
some of the proteases.
Formaldehyde, hydrocyanic acid, and soluble fluorides usually inhibit both
the activity of a cell and of the enzymes which it contains; while other
antiseptics, such as toluene, xylene, etc., prevent the growth of the cell,
or organism, without interfering with the activity of the enzymes which may
be present. By the use of this latter type of antiseptics, it is possible
to distinguish between chemical changes which are involved in the actual
development of a cell and those which can be brought about in other media
by means of the enzymes which are contained in the cell.
Any substance which increases the catalytic activity of an enzyme is known
as an "accelerator," or "activator"; while one which prevents this activity
is called an "inhibitor," or "paralyzer."
A type of accelerating influence quite different from that of electrolytes
is found in the effect of certain amino-acids upon enzyme action. The
influence of small amounts of asparagine in enormously increasing the
hydrolytic effect of amylase is an example. There is no known explanation
for this type of activation of the enzyme.
The influence of activators, or inhibitors, in providing favorable or
unfavorable conditions for the action of an enzyme, should not be confused
with the relation to the enzyme itself of what are known as "coenzymes" and
"antienzymes," discussed in the following paragraph.
COENZYMES AND ANTIENZYMES
In the cases of many enzymes of animal tissues, it has been found that they
are absolutely inactive unless accompanied by some other substance which is
normally present in the gland, or protoplasm, which secretes them. Thus,
the bile salts are absolutely necessary to the activity of trypsin, in its
characteristic protein-splitting action. Such substances are known as
"coenzymes." They can usually be separated from their corresponding enzymes
by dialysis, the coenzyme passing through the parchment membrane. Such
coenzymes are not killed by boiling the dialyzate, and the activity of the
enzyme is restored by adding the boiled dialyzate to the liquid which
remains within the dialyzer.
The best known example of a coenzyme in plant tissues is in connection with
the activity of the zymase of yeast cells. If yeast juice be filtered
through a gelatin filter, the colloidal enzymes which are left behind are
entirely inactive in producing fermentation, but may be restored to
activity again by mixing with the filtrate. An examination of this
filtrate, which contains the coenzyme for zymase, shows that it contains
soluble phosphates and some other substance whose exact nature has not yet
been determined, both of which are necessary to the activity of the zymase.
The phosphates seem to enter into some definite chemical combination with
the substrate sugars, while the other coenzyme seems to be necessary in
order to make possible the final breaking down of the sugar-phosphate
complex by the zymase. This phenomenon of coenzyme relationship is not very
frequently observed in plant enzyme studies, probably because the coenzyme
(if there be such, in the case which is under observation) usually
accompanies the enzyme itself through the various processes of extraction
and purification of the material for study. However, care must be taken in
all cases when dialysis is employed, to see that a possible coenzyme is not
separated from an otherwise active preparation.
An entirely different type of phenomenon is that exhibited by
"antienzymes." These are found in the various intestinal worms which live
in the digestive tracts of animals; and prevent the digestive action of the
enzymes of the stomach and intestines upon these worms. Probably similar
"antienzymes" are located in the mucous linings of the intestinal tract
itself, and serve to prevent the auto-digestion of these organs by the
active enzymes with which they are almost continually in contact.
The difference between an antienzyme, which protects material which would
otherwise be subject to the attack of an enzyme, and an inhibitor, which
renders the enzyme itself inactive, is apparent.
So far as is known, however, no such substances as antienzymes are present
in plant tissues; although the question as to why the proteoclastic enzymes
which are elaborated by a given mass of protoplasm do not attack the
protoplasm itself, might well be raised.
ZYMOGENS
It is apparent that, since enzymes are produced by protoplasm for the
special needs of any given moment or stage of development, there must be a
preliminary stage, or condition, in which they do not exert their
characteristic catalytic effect. When in this stage, the compound is known
as "proenzyme," or "zymogen." In this stage, it is inactive, but can be
made to exhibit its catalytic effect, usually by bringing it into contact
with a suitable activator. When once so activated, however, it cannot be
returned again to the inactive state.
This phenomenon has been studied in connection with the zymogens of the
digestive proteases, pepsin and trypsin. Trypsinogen may be rendered active
by contact with either calcium salts or with another substance (apparently
itself an enzyme) known as enterokinase, which is secreted in the
intestinal tract.
Similarly, proenzymes have been reported as occurring in numerous plant
tissues. These proenzymes are believed to be present in the plant cells in
the form of definite characteristic granules, which may be observed under
the microscope, and which disappear when the enzyme becomes active. Thus,
"proinulase" has been reported as occurring in artichoke tubers:
"prolipase," in castor beans; "proinvertase," in several species of fungi;
and, probably, "prooxidase," in tobacco leaves. In the case of the
last-named zymogen, it has been observed that after the zymogen has been
once activated, as in response to the need for increased activity due to
the entrance of the germs of certain leaf-diseases, it can once again
produce a second supply of the enzyme, but the process cannot again be
repeated.
Calcium salts, or very dilute acids, are usually energetic activators of
proenzymes.
PHYSIOLOGICAL USES OF ENZYMES
There can be no doubt that enzymes exert a tremendously important influence
in vital phenomena, by determining the rate at which the chemical changes
which are involved in these phenomena shall proceed. Since they do not
initiate reactions, and since they may catalyze reversible reactions in
either direction, it cannot be said that they determine the type of
reactions which will take place in any given mass of protoplasm; but,
undoubtedly, they do exert a determining influence upon the rate at which
the reaction will proceed, after the protoplasmic activity has determined
the direction in which it shall go.
Without the intervention of these catalyzing agents, it would be impossible
for reactions between these non-ionized organic components of the cell
contents to come to completion with anything like the marvelous rapidity
with which these changes must take place in order to permit the organism to
grow, to perform its necessary vital functions, or to adjust itself to the
changes in its environmental conditions.
Since the number of different reactions which take place within a living
cell is very great, and since these chemical changes are extremely variable
in type, it follows that the number of different enzymes which must exist
in either a plant or an animal organism is likewise very large. For
example, fourteen different enzymes have been isolated from the digestive
system, and at least sixteen from the liver, of animals. They are
universally present in living protoplasm of every kind, from the most
minute bacterium to the largest forest trees, in the plant kingdom; and
from the am[oe]ba to the whale, in animals.
While there is a great variety of enzymes which may be produced by a single
individual organism, the same enzyme may be found in the greatest variety
of organisms; as, for example, the protease trypsin, which has been found
in several species of bacteria, in the carnivorous plant known as "Venus'
Fly Trap," and in the human pancreas, as well as that of all other animals.
FURTHER STUDIES NEEDED
From the discussions which have been presented in this chapter, it is
apparent that the enzymes play a tremendously important part in vital
phenomena, by controlling the rate at which the biochemical reactions take
place in the cells of the living organism.
The means by which the protoplasm elaborates these all-important chemical
compounds are as yet absolutely unknown. Even the nature of the enzymes
themselves is still a matter of speculation and study. Much intensive study
is needed and should be given to these matters, for the purpose of
elucidating the methods by which the enzymes accomplish their remarkable
catalytic effects, and, if possible, the actual chemical nature of the
enzymes themselves. It is conceivable, of course, that if the latter object
of these studies should ever be reached, it might be possible to synthetize
enzymes artificially, and so to develop a means for the artificial
duplication of the synthesis of organic compounds with the same velocity
that this is done in the plant cells. Such a result would have a scientific
interest fully as great as did Wöhler's artificial synthesis of urea, which
proved that there is no essential difference in character between the
compounds which are the products of living organisms and those which are
produced in the laboratory; and, at the same time, might have an immensely
more important practical bearing, since it would lead the way to the
artificial production of the carbohydrates, proteins, fats, etc., for which
we are now dependent upon plant growth as the source of these materials for
use as human food.
References
BAYLISS, W. M.--"The Nature of Enzyme Action," 186 pages, _Monographs_ on
Biochemistry, London, 1919 (4th ed.).
EULER, H., trans. by POPE, T. H.--"General Chemistry of the Enzymes," 319
pages, 7 figs., New York, 1912.
EFFRONT, J., trans. by PRESCOTT, S. C.--"Enzymes and their
Application,--Enzymes of the Carbohydrates," 335 pages, New York, 1902.
EFFRONT, J., trans. by PRESCOTT, S. C.--"Biochemical Catalysts in Life and
Industry--Proteolytic Enzymes," 763 pages, New York, 1917.
GREEN, J. R.--"The Soluble Ferments and Fermentation," 512 pages,
Cambridge, 1901, (2d ed.).
GRUS, J.--"Biologie und Kapillaranalyse der Enzyme," 227 pages, 58 figs., 3
plates, Berlin, 1912.
HARDEN, A.--"Alcoholic Fermentation," 156 pages, 8 figs., Monographs on
Biochemistry, London, 1914.
PLIMMER, R. H. A.--"The Chemical Changes and Products Resulting from
Fermentations," 184 pages, London, 1903.
OPPENHEIMER, C., trans. by MITCHELL, C. A.--"Ferments and their Actions,"
343 pages, London, 1901.
CHAPTER XV
THE COLLOIDAL CONDITION
Reference has frequently been made, in preceding chapters, to the fact that
proteins, enzymes, lipoids, etc., exist in the protoplasm of plants and
animals in the colloidal condition. The properties and uses of these
compounds by plants depend so much upon this fact that, before proceeding
to the consideration of the actual physical chemistry of protoplasm itself,
it will be appropriate and profitable to give some attention to the nature
and significance of the colloidal condition of matter and of some of the
phenomena which grow out of it.
Every discussion of the colloidal condition in general properly begins with
reference to the work of the English physicist, Thomas Graham, who carried
on his investigations of the so-called "colloids" through a period of forty
years, beginning with 1851. His most important results were published,
however, from 1861 to 1864. Graham studied the diffusibility of substances
in solution through the parchment membrane of a simple dialyzer. As a
result of his earlier investigations, he divided all the chemical compounds
which were known to him into two groups, which he called "crystalloids" and
"colloids," respectively, the first including those substances which
readily diffused through the parchment membrane and the second those which
diffused only very slowly or not at all. He at first thought that
crystalloids are always inorganic compounds, while colloids are of organic
origin. He soon learned, however, that this distinction in behavior is not
always related to the organic or inorganic nature of the compound. He
further discovered that the same individual chemical element or compound
may exist sometimes in crystalloidal, and sometimes in colloidal, form.
This latter discovery led to the conclusion that diffusibility depends upon
the _condition_, rather than upon the _nature_, of the material under
observation.
As a result of the long series of investigations which were stimulated by
Graham's work, the modern conception is that diffusibility is a
_condition_ of matter when in minute subdivision, or in solution, in some
liquid, as contrasted with its _state_, or condition, when existing alone.
That is, the _state_ of a substance may be either gaseous, liquid, or
solid; and its _condition_ when in solution may be either crystalloidal or
colloidal. Substances which are in crystalloidal form, in true solution,
exist there in molecular or ionized condition; but, as will be pointed out
below, when in the colloidal condition they exist in aggregates which are
somewhat larger than molecules, but not large enough to be visible as
individual particles under the ordinary microscope, even under the highest
magnification which has yet been obtained. Colloidal particles are,
however, generally visible under the Zigmondy "ultramicroscope." (See
below.)
The use of the word "colloid" as a noun, or as the name for a substance
which is in the colloidal condition, is of the same nature as the use of
the words "gas," "liquid," and "solid," in such statements as "ice is a
solid," "water is a liquid," or "steam is a gas," etc.; i.e., the noun
represents a state or condition rather than an actual object or thing.
Hence, the expression "enzymes are colloids," means only that enzymes exist
in the colloidal condition, and not that enzymes represent a definite type
of substances having the group name "colloids."
THE COLLOIDAL CONDITION A DISPERSION PHENOMENON
When one substance is distributed through the mass of another substance,
the mixture is said to be a "two-phase system," composed of the _dispersed
phase_, or substance, and the _dispersion medium_, or _continuous_ phase,
through which the other substance is distributed. The following examples
illustrate the possibilities of such two-phase systems:
(1) Dispersion medium a gas.
(_a_) Disperse phase a liquid--mist in the air.
(_b_) Disperse phase a solid--smoke or dust in air.
(2) Dispersion medium a liquid.
(_a_) Disperse phase a gas--foams.
(_b_) Disperse phase a liquid--emulsions.
(_c_) Disperse phase a solid--suspensions.
(3) Dispersion medium a solid.
(_a_) Disperse phase a gas--solid foams, pumice stone, etc.
(_b_) Disperse phase a liquid--liquid inclusions in minerals.
(_c_) Disperse phase a solid--alloys, colored glass, etc.
Although the same general principles of physical chemistry apply to all
two-phase systems, the term "colloidal condition" is commonly used only in
connection with a particular type of dispersions, in which the dispersion
medium is a liquid and the dispersed material is either a solid or a
liquid.
Thorough and careful studies have shown that when a solid or a liquid is
introduced into another liquid, and becomes dispersed or distributed
through it, the mixture may be either a true solution, a colloidal
solution, or a mechanical suspension. The characteristic differences
between these three conditions may be tabulated as follows: although the
significance of some of the phrases used will not be apparent until the
phenomena in question have been considered in some detail.
-----------------------+------------------------+------------------------
True Solutions. | Colloidal Solutions. | Suspensions.
-----------------------+------------------------+------------------------
| |
(_a_) Particles of the | |
disperse phase are: | |
| |
In molecular | In colloidal | In mechanical
subdivision | subdivision | subdivision
| |
Invisible | Visible under | Visible under
| "ultrascope" | microscope or to naked
| | eye
| |
Less than 1µµ | 1µµ to 1µ in diameter | Greater than 1µ in
in diameter[6] | | diameter
| |
Pass through filters | Pass through filters | Do not pass through and
parchment membranes | but not through | filters or parchment
| parchment |
| |
In molecular motion | In Brownian movement | In gravitational
| | movement
| |
(_b_) The system | |
exhibits: | |
| |
High osmotic pressure | Low osmotic pressure | No osmotic pressure
| |
Transparency | "Tyndall phenomenon" | Is generally opaque
| |
No gel-formation | Forms gels | No gel-formation
| |
-----------------------+------------------------+------------------------
FOOTNOTES:
[6] 1µ is one-thousandth of a millimeter; 1µµ is one-thousandth of a µ, or
one millionth of a millimeter.
It is recognized by all students of these matters that it is not possible
to draw a sharp dividing line between these three types of conditions, and
that they shade into each other, in many cases; but in general it may be
said that a colloidal solution is one in which the dispersed particles are
usually between 5µµ and 200µµ in diameter, are difficultly or not at all
diffusible through the membrane of a simple dialyzer, cannot be filtered
out of solution, do not settle out under the action of gravitation, and are
visible only under the "ultramicroscope"; and one which has certain
peculiar optical, osmotic, and other physical and chemical properties.
Since colloidal particles are very minute in size, they possess very large
relative surface areas as compared with their total mass or volume, very
high surface tension, and a relatively high surface energy as compared with
their total, or molecular, energy. These properties bring into play, in a
substance which is in the colloidal condition, in a remarkable degree, all
the phenomena which are associated with surface boundaries between solids
and liquids, liquids and gases, etc.
The properties arising out of the colloidal condition are of such
tremendous importance in connection with the vital phenomena exhibited by
cell protoplasm that it is necessary to give some detailed consideration to
them here. Many large volumes dealing with this condition of matter have
been written, and it is very difficult to condense even the most important
facts concerning it into a few pages, but an attempt has been made to
present in this brief summary the most essential facts and principles
involved in the colloidal phenomena.
NOMENCLATURE AND CLASSIFICATION
Colloidal mixtures may exist in two different forms: one, in which the
mixture is fluid and mobile, like a true solution, is known as a "sol"; and
the other, which is a semi-solid, or jelly-like, form, is known as a "gel."
Sols may be easily converted (or "set") into gels, by changes of
temperature or of the electrolyte content, or by changes in the
concentration of the mixture, etc., and in most cases gels can be converted
again into sols. In some cases, however, gel-formation is irreversible, the
gels are permanent and cannot be changed back again into sols by any known
change in environmental conditions.
Depending upon whether the liquid dispersion medium is water, alcohol,
ether, etc., sols are known as "hydrosols," "alcosols," "ethersols," etc.;
and gels as "hydrogels," "alcogels," etc.
Sols in which the disperse phase is a solid are known as "suspensoids";
while those in which it is a liquid are "emulsoids." Thus, sols of most
inorganic compounds, of dextrin, gelatin, and (probably) of casein, etc.,
are suspensoids; while sols of egg-albumin, of oils, etc., are emulsoids.
The classification of these substances into suspensoids and emulsoids is,
however, more a matter of convenience than of real difference in
composition, since it is practically impossible to say whether many of the
organic substances which normally exist in colloidal form are themselves
liquids or solids, when in the non-dispersed form.
CONDITIONS NECESSARY TO THE FORMATION OF SOLS
Suspensoids differ from mechanical suspension of solids in a liquid in that
in the latter the solid particles settle toward the bottom of the mixture,
because of the effect of the attraction of gravity upon them. The rate at
which such particles settle depends upon the size and density of the
particle and the viscosity of the liquid, and can be roughly calculated
from the formula for Stokes' law for the rate of falling of a spherical
body in a liquid. This formula is
_V_ = 2_r_^2(_s_ - _s_´)_g_ / 9_n_;
_V_ = velocity of the falling body, in millimeters per second;
_r_ = radius of the particle, in millimeters;
_s_ = specific gravity of the solid;
_s_´ = specific gravity of the liquid;
_g_ = the attraction of gravity, in dynes;
_n_ = the viscosity of the liquid.
For example, if this formula be applied to determine the rate at which the
particles of gold of the size of those in a red gold sol would settle, if
they were in mechanical suspension in water (_r_ = 10µµ, or
one-ten-thousandth of a millimeter; _s_ = 19.3; _s_´ = 1; _g_ = 980, and
_n_ = 0.01), it will be found that such particles will settle at the rate
of approximately 0.0146 millimeter per hour, or a little over 10 mm. (0.4
inch) per month. Hence, the settling of such particles, if in mechanical
suspension, would be measurable, although very slow. Shaking up the
_suspension_ would cause the particles to rise through the liquid again.
But in a gold sol, or _suspensoid_, which contains particles of gold of the
size used in this calculation, the gold particles do not settle, even at
the slow rate as calculated above. They remain uniformly distributed
throughout the liquid for an indefinite period or time. The reason for
this phenomenon undoubtedly lies in the fact that these minute particles
carry an electric charge, which, is of the same sign for all of the
particles and results in a repellent action which keeps the particles in
constant motion. This constant motion may easily be conceived to keep the
particles uniformly distributed throughout the liquid, just as constant
shaking would keep those of a mechanical suspension uniformly distributed
through the mixture.
The sign of the electric charge on the particles of a sol may be either
negative or positive, depending upon the chemical nature and dielectric
constants of the two phases of the system. The proportion of the total
electric charge of the system which is of the opposite sign to that borne
by the dispersed particles is, of course, borne by the liquid which
constitutes the other phase. The origin of this electric charge on the
colloidal particles is, as yet, not known with certainty; but it seems
probable that it is due to a partial ionization of these small particles,
similar to, but not so complete as, that which takes place when compounds
which are soluble go into true solution in water, or other solvents which
bring about the dissociation of dissolved substances.
The conditions necessary to bring a solid substance into a colloidal
mixture with some liquid, or, in other words, to produce a suspensoid sol,
require that the proportion of liquid to solid shall be large and some
means of disintegrating the material which is to be dispersed into very
fine particles. Many common chemical reactions, if carried out in very
dilute solutions, result in the production of sols, especially if a small
amount of some emulsoid is present in the reacting mixture; sols produced
in this way are very stable, and the emulsoid which is used in stabilizing
the sol is known as a "protective colloid." Direct methods of
disintegration; such as reduction by chemical agents, discharge of a strong
electrical current through the substance which is to be dispersed while it
is submerged in the liquid, alternate treatment of finely ground material
with alkali and acid so as to frequently change the electric charge, etc.,
are utilized for bringing inorganic compounds into the colloidal state.
Suspensoids usually contain less than 1 per cent of the solid dispersed
through the liquid. In fact, extreme dilution is one of the necessary
conditions for suspensoid-formation.
Emulsoids are much more easily produced than are suspensoids. The property
of forming an emulsoid seems to be much more definitely a characteristic of
the substance in question than does the formation of sols from solids
which, under other conditions, may form true solutions. This difference may
be due to the fact that the liquids which easily form emulsoids (usually
those of organic origin) have very large molecules, so that the transfer
from molecular to colloidal condition involves much less change in such
cases than it does in the case of solid (inorganic) substances of
relatively low molecular weight. This view of the matter is further borne
out by the fact that solids which have very large molecules (generally of
organic origin) take on the colloidal form much more readily than do those
of small molecular size.
At the same time, a given liquid may form a true emulsoid when introduced
into one other liquid and a true solution when introduced into another.
Thus, soaps form emulsoids with water (true hydrosols); but dissolve in
alcohol to true solutions, in which they affect the osmotic pressure, the
boiling point of the liquid, etc., in exactly the same way that the
dissolving of other crystalloids in water affects the properties of true
aqueous solutions. Again, ordinary "tannin," when dissolved in water,
produces a sol, which froths easily, is non-diffusible, etc.; but when
dissolved in glacial acetic acid, it produces a true solution.
The concentration of the disperse phase may be much greater in the case of
emulsoids than it can be in suspensoids. This is probably because the
dispersed particles do not carry so large an electric charge and are not in
such violent motion.
GEL-FORMATION
The one property which most sharply distinguishes sols from true solutions
is their ability to "set" into a jelly-like, or gelatinous semi-solid,
mass, known as a "gel," without any change in chemical composition, or
proportions, of the two components of the system. In the gel, the two
components are still present in the same proportions as in the original
sol; but the mixture becomes semi-solid instead of fluid in character.
Thus, an agar-agar sol containing 98 per cent of water sets into a stiff
gel; while many other gels which contain 90 to 95 per cent of water can be
cut into chunks with a knife and no water will ooze from them. The water is
not in chemical union with the solid matter in the form of definite
chemical hydration, however, as the same gel is formed with all possible
variations in the water content.
Gels may be either rigid, as in the case of those of silicic acid, etc., or
elastic, as are those of gelatin, egg-albumin, agar-agar, etc. The latter
are the common type of gels among organic colloids. They can be easily
changed in shape, or form, without any change in total volume.
In gel-formation, the two phases of the system take a different
relationship to each other. The disperse, or solid, phase becomes
associated into a membrane-like, or film, structure, surrounding the liquid
phase in a cell-like arrangement. That is, the whole mass takes on a
structure similar to a honeycomb except that the cells are roughly
dodecahedral in shape, instead of the hexagonal cylinders in which the bees
arrange their comb cells, in which the original disperse phase constitutes
the cell-walls and the original liquid, or continuous phase, represents the
cell-contents. The cells of an elastic gel resemble closely the cells of a
plant tissue in many of their physical properties. They are roughly
twelve-sided in shape, as this is the form into which elastic spherical
bodies are shaped when they are compressed into the least possible space.
=Imbibition and Swelling of Gels.=--When substances which are natural gels,
such as gelatin, agar-agar, various gums, etc., are submerged in water,
they imbibe considerable quantities of the liquid and the cells become
distended so that the mass of the material swells up very considerably.
This swelling will take place even against enormous pressures. For example,
it has been found that the dry gel from sea-weeds will swell to 330 per
cent of its dry volume, if immersed in water under ordinary atmospheric
pressure; but that it will increase by 16 per cent of its own volume when
moistened, if under a pressure of 42 atmospheres.
During the swelling of gels by imbibition of water, the total volume of the
system (i.e., that of the original dry gel plus that of the water absorbed)
becomes less. For example, a mixture of gelatin and water will, after the
gelatin has swelled to its utmost limit, occupy 2 per cent less space than
the total volume of the original gelatin and water. It has been computed
that a pressure equivalent to that of 400 atmospheres would be necessary to
compress the water to an extent representing this shrinkage in volume.
On the other hand, gels when exposed to the air lose water by evaporation,
shrink in volume, and finally become hard inelastic solids, as in the case
of the familiar forms of glue, gelatin, agar-agar, gum arabic, etc.
The difference in the relation of gels and that of non-colloidal solids to
water may be illustrated by the different action of peas, beans, etc., and
of a common brick, when immersed in water. Each of these substances, under
these conditions, absorbs, or "imbibes," water; but the peas and beans
swell to more than twice their original size and become soft and elastic,
while the brick undergoes no change in size, elasticity, or ductility. In
all cases of colloidal swelling, the swollen body possesses much less
cohesion, and greater ductility, than it had before swelling. The essential
difference in the two types of imbibition is that in the case of the
non-swelling substances the cohesion, or internal attraction of the
molecules of the material, is too great to permit them to be forced apart
by the water; while in colloidal swelling, the particles are forced apart
to such an extent as to make the tissue soft and elastic. It is possible,
of course, to make this separation go still further, until there is an
actual segregation of the molecules, when a true solution is produced; for
example, gum arabic when first treated with water swells into a stiff gel,
then into a soft gel, and finally completely dissolves into a true
solution.
=Reversibility of Gel-formation.=--In some cases, the change of a sol to a
gel is an easily reversible one. Glue, gelatin, various fruit jellies,
etc., "melt" to a fluid sol at slightly increased temperatures and "set"
again to a gel on cooling, and the change can be repeated an indefinite
number of times. On the other hand, many gels cannot be reconverted into
sols; that is, the "gelation" process is irreversible. For example,
egg-albumin which has been coagulated by heat cannot be reconverted into a
sol; casein of milk when once "clotted" by acid cannot again be converted
into its former condition, etc. Irreversible gelation is usually spoken of
as "coagulation." Some coagulated gels, by proper treatment with various
electrolytes, etc., can be converted into sols, the process being known as
"peptization"; but in such "peptized" hydrosols, the material usually
exists in a different form than originally, having undergone some chemical
change during the peptization, and the coagulation and peptization cannot
be repeated, that is, the process is not a definitely reversible one.
=Importance of Gel-formation.=--From the physiological point of view,
gel-formation is undoubtedly the most important aspect of colloidal
phenomena. In the first place, the ability to absorb and hold as much as 80
to 90 per cent of water in a semi-solid structure is of immense
physiological importance. In no other condition can so large a proportion
of water, with its consequent effect upon chemical reactivity, be held in a
structural, or semi-solid, mass. But a vastly more significant feature of
the conditions supplied by the gel lies in the fact that the non-water
phase, or phases, of the system are spread out in a thin film, or membrane,
thus giving it enormous surface as compared with its total volume. This
effect is easily apparent if one thinks of the enormous surface which is
exposed when a tiny portion of colloidal soap is blown out into a
"soap-bubble" several inches in diameter. This condition brings into play
all the phenomena resulting from surface boundaries between solids and
liquids, liquids and liquids, liquids and gases, etc., from surface
tension, surface energy, etc. Among these effects may be cited those of
adsorption, increased chemical reactivity due to enlarged areas of contact,
permeability and diffusion, etc., the importance of which in the vital
phenomena of cell-protoplasm will be discussed in detail in the following
chapter.
GENERAL PROPERTIES OF COLLOIDAL SOLUTIONS
=Non-diffusibility.=--The most characteristic property of all sols is the
failure of the suspended particles to pass through a parchment, or any
similar dialyzing membrane.
=Visibility under the "Ultramicroscope."=--The particles of a sol, in
contrast with the molecules of a true solution, are visible as bright
scintillating points under the ultramicroscope. This is a modification of
the type of dark-field illumination of the ordinary microscope, as applied
to microscopic studies, in which the solution to be studied is contained in
a small tube or box of clear glass which is mounted on the stage of an
ordinary microscope and instead of being illuminated from below by
transmitted light is illuminated by focusing upon it the image of the sun,
or of some other brilliant source of light such as an electric arc, by
passing the rays from the source of light through a series of condensing
lenses which are adjusted at the proper distance and angles to bring the
image of the illuminating body within the tube containing the substance
which is to be examined and in the line of vision of the microscope.
Obviously, this results in intense illumination of any particles in the
solution which come within this brilliant image of the sun, or arc, and
therefore renders visible particles which are of less diameter than the
wave-length of ordinary light (450µµ to 760µµ for the visible spectrum)
and, hence, are not visible by the ordinary means of illumination in the
direct line of vision. It will be apparent that what is seen in the field
of the ultramicroscope is not the particles themselves, but rather the
image of the sun (or other illuminating body) falling upon the particles
which come within the image, just as one does not see the paper but only
the image of the sun when the rays from the sun are brought to a focus upon
a sheet of paper through any ordinary convex lens, or "burning glass."
Hence, the ultramicroscope gives no idea of the shape, color, or size of
the particles upon which the image falls; but it does permit the counting
of the number of particles within a given area, and a study of their
movements, from which it is possible, by mathematical computations, to
calculate the relative size of the particles themselves. Repeated studies
have shown that particles of the sizes between 5µµ and 250µµ in diameter,
which are visible under the ultramicroscope, are sufficiently small to
bring about the surface phenomena which are known as properties of
colloidal solutions. Further, the ultramicroscope permits the observation
of the growth, or disintegration, under various chemical reagents, of the
individual colloidal particles, which appear as scintillating points in the
field of the microscope; and the study of changes in relationships during
gel-formation, peptization, etc.
=The "Tyndall Phenomenon."=--Colloidal solutions exhibit this phenomenon;
that is, if a bright beam of light be passed through a sol which is
contained in a clear glass vessel having parallel vertical sides, and the
solution be viewed from the side, it appears turbid and often has a more or
less bluish sheen. This effect is due to the small particles in the sol, of
polarizing the light which is reflected from them, the blue rays being bent
more than are those in the other part of the spectrum. The Tyndall
phenomenon is similar in its effect in making the tiny particles of the sol
visible to the illumination of the dust particles in the air of a darkened
room when a ray or narrow beam of light passes through it. In a true
molecular solution, the particles are too small to be visible by this mode
of illumination.
=Other Optical Properties.=--Sols are generally translucent and opalescent;
many of them are highly colored, some of the sols of gold, platinum and
other heavy metals possessing particularly brilliant colors. In general,
metallic suspensoids are red, violet, or some other brilliant color; while
inorganic suspensoids are bluish white, and emulsoids generally blue to
bluish white.
=Formation of Froth, or Foam.=--Colloidal solutions, especially those of
the natural proteins, fats, glucosides, gums, and the artificial soaps,
have a strong tendency to produce froth, or foam, when shaken; this being
due to the enormous surface tension resulting from the finely divided
condition of the dispersed material.
=Low Osmotic Pressure.=--All colloidal solutions exhibit a very low osmotic
pressure; the freezing point of the dispersion medium is lowered only very
slightly and its boiling point is only very slightly raised by the presence
of the dispersed particles in it.
=Precipitation by Electrolytes.=--Sols of all kinds are precipitated, or
caused to form gels, by the addition of electrolytes, since these cause a
disturbance of the electric charge on the dispersed particles, to which the
colloidal condition is due. In the case of most emulsoids and of a few of
the suspensoids, this change converts the mass into a stiff gel; but in
that of many of the metallic suspensoids, the dispersed particles are
gathered together into larger aggregates, which settle out of the liquid in
the form of a gelatinous precipitate. In the latter case, the effect is
usually spoken of as "precipitation" by electrolytes; while in the former,
it is called "coagulation," or "gelation."
The effectiveness of the various electrolytes in bringing about this change
is proportional to their valency; bivalent ions are from 70 to 80 times,
and trivalent ions about 600 times as effective as monovalent ions.
Further, all sols in which the dispersed particles carry a charge of the
opposite sign likewise precipitate both suspensoids and emulsoids.
A demonstration of the presence of an electric charge on the particles of a
sol and a determination of its sign can be made by placing the solution in
a U tube, with a layer of distilled water above the sol in each arm of the
tube, and then passing an electric current through the contents of the
tube, keeping the electrodes in the distilled water, so that the migration
of the particles toward one pole or the other can be observed by their
appearance in the clear water at that end of the tube; or by passing an
electric current through the observation chamber of an ultramicroscope, in
which the solution under examination has been placed, and observing the
migration of the particles across the field toward either one or the other
(positive or negative) electrode.
_Emulsoids and suspensoids_ differ in their properties in the following
respects. Suspensoids are always very dilute, containing less than 1 per
cent of the dispersed solid; while emulsoids may be prepared with widely
varying proportions of the two component liquids. Suspensoids have a
viscosity which is only slightly greater than that of the liquid phase when
it exists alone, and their viscosity varies with the proportion of
dispersed solid which is present in the sol; while emulsoids have a very
high viscosity in all cases. Emulsoids usually form stiff gels when treated
with electrolytes; while suspensoids more commonly yield gelatinous
precipitates under the same conditions.
Suspensoids and emulsoids which carry electric charges of opposite sign
mutually precipitate each other. But emulsoids often protect suspensoids
from precipitation by electrolytes, by forming a protective film around the
particles of the suspensoids, which prevents the aggregation of the
particles into the precipitate form.
ADSORPTION
If a sol be precipitated or coagulated by the action of an electrolyte,
substances which may be present in solution in the liquid of the sol are
carried out of solution and appear in the gel or precipitate. This
phenomenon is known as "adsorption," which means the accumulation of one
substance or body upon the surface of another body, as contrasted with
"absorption," which means the accumulation of one substance within the
interior of another. Since substances which are in the colloidal form have
very large relative surface areas, it follows that the opportunity for
surface adsorption on colloidal materials is very great.
Surface adsorption is a common phenomenon. It was extensively studied by
the physicist, Willard Gibbs, who showed that adsorption will take place
whenever the surface tension of the adsorbing body will be lowered by the
concentration in its surface layer of the material which is available in
the solution or other surrounding medium.
As applied to colloidal phenomena, adsorption may be exhibited in either
one of four different ways, as follows: (1) A crystalloidal substance which
is in solution may be adsorbed on the colloidal particles of a hydrosol, so
that if the mixture be dialyzed, or filtered through a so-called
"ultrafilter" (i.e., a filter with pores so small that it will retain
colloidal particles) the dissolved crystalloid will remain with the
separated colloidal particles, or the dissolved crystalloid will not react
chemically as it would in a free solution. For example, if to a solution of
methylene blue, which dyes wool readily, there be added a small quantity of
albumin (a colloidal substance), the dye is adsorbed by the albumin and
will no longer color wool with anything like the same readiness. (2) During
gel-formation, electrolytes and other soluble substances which may be
present in solution in the liquid may adsorbed out of the solution and
appear in the gel. For example, a precipitate of aluminium hydroxide, or of
silicic acid, is nearly always contaminated with the soluble salts which
are present in the solution, and can be prepared in pure form only by
repeated filtering, redissolving, and reprecipitating. (3) Colloidal
substances may be removed from sols by being adsorbed upon porous materials
like charcoal, fuller's earth, hydrated silicates, etc. For example, animal
charcoal (or bone black) is used commercially for the clarification of
sugar solutions, because it adsorbs out of these solutions the colloidal
proteins, coloring matters, etc., with which they are contaminated. (4)
Finally, colloids mutually adsorb each other, as in the case of the
"protective colloids" previously referred to.
Certain characteristics of adsorption phenomena are of interest and
importance from both the physiological and the industrial point of view.
The following may be mentioned: (_a_) _Amount of adsorption._ Relatively
more material is adsorbed out of dilute solutions than out of more
concentrated ones. An increase of ten times in the concentration of the
dissolved material results in only four times as much adsorption by the
colloidal substance which may be introduced into the two solutions. In
this, adsorption differs from chemical action, as the latter is
proportional to the concentration of the reacting material which is present
in the solution. (_b_) _Adsorption out of different liquids_, by the same
adsorbing body, is different in amount. It is usually greatest out of
water. Hence, many dyes may be adsorbed out of water by charcoal, porous
clay, etc., and if the latter be then introduced into alcohol, or ether,
the dye goes back into solution in these latter liquids. This process is
often used industrially and in the laboratory for the purification of such
substances when they are present in impure form in aqueous solutions. (_c_)
_Selective adsorption._ Different substances are not adsorbed out of the
same solvent to the same extent by the same adsorbing agent. Advantage is
taken of this fact when filter paper is used in the so-called "capillary
analysis" to separate different dyes, or other colloidal materials which
have been stained different colors, into alternate layer by reason of the
different rate at which the paper adsorbs the different materials out of
the solution in which they are present together. (_d_) _Similar relative
adsorption by different adsorbing agents._ Although different adsorbing
agents may possess varying active surfaces and hence, variable adsorbing
power, or rates of adsorption, they adsorb the same relative amounts of
different materials; i.e., if substance _A_ adsorbs more of _X_ than it
does of _Z_ out of any given solution, substance _B_ will likewise adsorb
more of _X_ than of _Z_ out of the same solution; although the actual
amounts adsorbed by _A_ may be quite different from those adsorbed by _B_.
CATALYSIS AFFECTED BY THE COLLOIDAL CONDITION
The velocity of a chemical reaction is the net result of opposing
influences. It is directly proportional to the chemical affinity of the
reacting bodies and inversely proportional to the so-called "chemical
resistance." The first factor, chemical affinity, is not easily measured,
as it depends upon both the mass of the reacting molecules, atoms, or ions,
and their attraction for each other. But if, as the result of chemical
affinity, a reaction takes place, it is evident that the time required for
its completion (which measures the velocity of the reaction) is made up of
two separate periods. The first is the time required for the reacting
molecules to come into contact; and the second is that required for the
molecular rearrangement which constitutes the reaction. Clearly, the time
required for the substances to come into molecular contact will be greatly
diminished if they are mutually adsorbed in large quantities on the
extended surface area of some colloidal catalyst which is present in the
mixture rather than scattered throughout its entire volume. The application
of this principle to the catalysis of hydrolytic reactions is not apparent,
if it is considered that the H_{2}O molecules which cause the hydrolysis
are those of the solvent itself; but is clear on the assumption (which is
discussed in the following chapter) that the water which enters into a
colloidal complex is in multimolecular form, represented by the formula
(H_{2}O)_{_n_}, in which the oxygen atoms are quadrivalent and, hence, much
more active chemically than as illustrated in the simple solvent action of
water.
Hence, the surface adsorption of reacting bodies by a colloidal catalyst
may have a very important influence in decreasing the time required to
bring the reacting molecules into intimate contact, and so increasing the
velocity of the reaction.
But the colloidal condition of the catalyst may also aid in decreasing the
"chemical resistance" which tends to slow up the reaction. Chemical
resistance may be understood to be the internal molecular friction of the
densely packed atoms within the reacting molecule, which tends to prevent
the molecular rearrangement and so to prolong the second period of the
reaction time. To overcome this friction and so decrease the reaction time,
some form of energy is necessary. If there be present in the solution in
which the reaction is taking place some colloidal catalyst, and if the
reacting bodies are concentrated at the surface boundaries between the two
phases of the colloidal system, they may be conceived to be within the
sphere of influence of the surface energy of the dispersed particles of the
catalyst, so that this may furnish the energy necessary to overcome the
chemical resistance of the reacting bodies, and so to speed up the second
portion of the reaction time.
From these considerations, it would appear that the colloidal condition of
such catalysts as enzymes, etc., has much to do with their ability to
increase reaction velocities, both by reducing the time necessary for the
reacting bodies to come into molecular contact and by furnishing the energy
to overcome the chemical resistance to the molecular rearrangement which
constitutes the reaction itself. Evidence in favor of the accuracy of this
view of the nature of the catalytic action of colloidal substances is
afforded by the facts that catalysts accelerate the velocity of reversible
reactions in either direction and that they do not change the point of
final equilibrium, in any case; that is, they do not affect the nature or
direction of the reaction, but only accelerate a chemical change which
would otherwise take place more slowly because of the stability (or
chemical resistance) of the molecules involved, or their inability to come
quickly into intimate molecular contact.
These facts and principles have been clearly established in many studies of
the nature of enzyme action (enzymes are typical colloidal catalysts) and
probably apply equally well to the action of other types of colloidal
catalysts. On the other hand, the catalytic action of certain inorganic and
non-colloidal substances, such as the action of acids in accelerating the
hydrolysis of carbohydrates, etc., may be conceived to be due to chemical
influences upon the internal molecular resistance, which are similar in
their effects, but entirely different in their mechanism, from the physical
effects of the surface boundary phenomena of the colloidal catalysts.
INDUSTRIAL APPLICATIONS OF COLLOIDAL PHENOMENA
Large numbers of industrial processes are based upon colloidal phenomena.
Many of these processes were known and practiced long before the nature of
the phenomenon itself was understood. But with the coming of the knowledge
of the nature, causes, and possibilities of the control, of the colloidal
condition of the materials involved, immense improvements in the economy of
the process, or the quality of the end-products, have been worked out, in
many cases. Many volumes of treatises concerning the industrial
applications of colloidal phenomena have been written. Any discussion of
these would be out of place here; but the following list of examples will
serve to illustrate the immense importance of these matters both in
industry and to the needs of everyday life: the tanning of leather; the
dyeing of fabrics; vulcanizing rubber; mercerizing cotton; sizing textile
fabrics; manufacture of mucilages and glues; manufacture of hardened casein
goods; manufacture of celluloid; production of colloidal graphite for
lubrication; the prevention of the smoke nuisance by electric deposition;
the purification of sewage; the manufacture of soaps; the manufacture of
butter, cheese, and ice cream; fruit jellies, salad dressings, etc. This
list could be extended to a great length, but is already long enough to
emphasize the very great importance and practical value of colloidal
phenomena in daily life.
NATURAL COLLOIDAL PHENOMENA
Many of the phenomena of nature are colloidal in character. These may be
observed in the mineral, the animal, and the vegetable kingdoms. Here,
again, a lengthy discussion of the nature of these phenomena would be out
of place in this connection, and a few typical examples will serve to
illustrate the general importance in nature of this property of matter.
In the soil, the following properties are easily recognizable as definite
colloidal phenomena: water-holding capacity of clays, silts, loams, etc.;
adsorption (or "fixation") of soluble plant foods so that they are not
readily leached out of the soil by drainage; flocculation and
deflocculation of clay, etc.
In the animal body; the contraction of muscles, the conveyance of nerve
stimuli, etc., are undoubtedly accomplished by colloidal changes; and the
existence of insoluble casein and fat in colloidal form in milk insures the
proper nourishment of the young of nearly all species of animals.
In both plants and animals, as will be pointed out in the following
chapter, practically all the vital activities of the cell protoplasm are
definite manifestations of colloidal phenomena. Enzymes perform their
catalytic functions by reason of their colloidal form. Proteins exist in
colloidal form and are the seat of all vital functions. The regulation of
the passage of materials into and out of the cell is governed by minute
changes in the electrolyte concentration, etc., which produce enormous
changes in the colloidal character of the protoplasm.
It is apparent, therefore, that the study of the colloidal condition of
matter and of the properties arising out of it is of immense importance to
the biochemist. No other single field is capable of yielding more fruitful
results to the plant physiologist, in his studies of the response of plants
to changes in their environment, or of the mechanism by which plants
perform their internal functions.
References
BECHHOLD, H., trans. by BULLOWA, J. G. M.--"Colloids in Biology and
Medicine," 463 pages, 54 figs., New York, 1919.
BURTON, E. F.--"The Physical Properties of Colloidal Solutions," 200 pages,
18 figs., London, 1916.
CASSUTO, L.--"Der Kolloide Zustand der Materie," 252 pages, 18 figs.,
Dresden and Leipzig, 1913.
LIESEGANG, R. E.--"Beiträge zu einer Kolloidchemie des Lebens," 144 pages,
Dresden, 1909.
OSTWALD, W., trans. by FISCHER, M. H.--"Theoretical and Applied Colloid
Chemistry," 218 pages, 43 figs., New York, 1911.
OSTWALD, W., trans. by FISCHER, M. H.--"A Handbook of Colloid-Chemistry,"
278 pages, 60 figs., Philadelphia, 1915.
TAYLOR, W. W.--"The Chemistry of Colloids," 328 pages, 22 figs., New York,
1915.
ZIGMONDY, R., trans. by ALEXANDER, J.--"Colloids and the Ultramicroscope,"
238 pages, 2 plates, New York, 1909.
ZIGMONDY, R., trans. by SPEAR, E. B.--"The Chemistry of Colloids," 274
pages, 39 figs., New York, 1917.
CHAPTER XVI
THE PHYSICAL CHEMISTRY OF PROTOPLASM
Thus far, we have considered the chemical nature of the various groups of
compounds which are found in the tissues of living organisms, laying
emphasis upon those which are of plant origin. These compounds constitute
the material, or machinery, of the cell, and their various transformations
furnish the energy for its operation. We come now to a study of the mode of
its operation, or the processes of vital phenomena.
Our knowledge of these matters is not yet far enough advanced to permit a
definite statement as to whether there is any difference between the
protoplasm of plant tissues and that of animal origin in their modes of
action, or in the physical-chemical changes which constitute the vital
phenomena in the two groups of living organisms. Thus far, no such
differences have been discovered. Hence, in the following discussions, no
attempt is made to differentiate between animal and plant protoplasm. Most
of the facts and principles which are here presented have been developed as
the result of the study of the physiological chemistry of animal life. No
similar careful study of plant chemistry has yet been carried out; but
preliminary studies seem to indicate that the same general principles apply
to all protoplasm, regardless of whether it is of plant or of animal
origin. It is possible, of course, that further studies of plant protoplasm
will render necessary some modifications of some of these views as applied
to the growth of plants; but they are believed to represent the best which
is now known of the physical chemistry of the plant-cell activities.
HETEROGENEOUS STRUCTURE OF THE CELL
Examination of cell protoplasm under the microscope reveals that it is not
a simple homogeneous mass. In the first place, it has a definite
structure, composed of (_a_) a nucleus; (_b_) numerous granular bodies of
different sizes and kinds; and (_c_) a clear mass of colloidal material,
which (if observed under the ultramicroscope, or photographed by
ultra-violet light) is apparently made up of very minute particles of many
different types of materials; the whole mass, in the case of plant
protoplasm, being generally surrounded by (_d_) a differentiated layer
known as the cell-wall. The actual internal structural arrangement of the
clear colloidal mass is uncertain; but its properties indicate that it may
be considered to be like a mass of foam (resembling a compact mass of
soap-bubbles) the compartments of the foam being, of course, very minute
and the films themselves almost infinitely thin, the contents of each
compartment being probably liquid, and the whole composing a typical
colloidal gel of complex composition.
This conception may not be accurate in every detail, but it seems to fit
very closely the conditions and reactions of cell protoplasm. Furthermore,
it is obvious that the definite structure, or form, of the cell is
essential to its life; since, if the structure be destroyed by any kind of
mechanical injury (freezing of the cell contents, resulting in the
puncturing of the membranes by ice crystals; rupturing of the films, or
cell-walls, by grinding with sharp sand, etc.) so as to bring about an
intermingling of the parts which are segregated from each other in the
organized structure, there results an immediate exhibition of abnormal
chemical actions, accompanied by the liberation of carbon dioxide, and the
death of the cell.
A proper mental picture of the organization of the cell structure and of
the interrelation of all its working parts is suggested by the figure of a
well-organized chemical factory, with the different chemical
transformations which are involved in the whole process being carried on in
different portions, or rooms, of the factory, with the various intermediate
and final products regularly and systematically transported from one room
to another as they are needed to keep each individual step in the whole
process going at the proper rate, and with the different parts of the whole
factory working in smooth coordination with each other. Any disturbance of
the mechanism in any particular room, or any abnormal condition which
breaks down the coordination or results in the mixing of the reagents or
processes of adjoining rooms in improper order or proportions, produces
instant destruction of the normal process, abnormal reactions take place,
and the factory output is interrupted.
No other conception than this one of a definite structure and coordination
of the different working parts of a cell can adequately account for the
great variety of chemical changes which are constantly going on in any
given cell. It is wholly inconceivable that a homogeneous mass of all the
varying chemical compounds which are contained in any given quantity of
protoplasm could either exist or produce any regular sequence of chemical
reactions. Structure, or organization of the cell-contents into separate
colloidal compartments, and the segregation of cell-contents into masses
having different functions, is essential to any reasonable conception of
how the cell performs its various activities.
The best understanding of the structural arrangement is afforded by the
conception that protoplasm consists of a colloidal gel, or sometimes a very
viscid sol, containing water, salts, carbohydrates, fats, proteins, and
enzymes. Evidence in favor of this conception is afforded by the appearance
of protoplasm under a high-power microscope, and by the close resemblance
of the processes which go on in it, and its responses to external stimuli,
to those of an artificial gel of similar chemical composition.
Two different conceptions of the form in which the chemical components
exist in this mass have been advanced. One is that they are in true
molecular unions, known as "biogens," and that the reactions which take
place in the mass may, therefore, be studied from the same basis as are
reactions between similar substances when they take place in a beaker or
test tube in the laboratory. It would seem, however, that the constantly
varying proportions of the materials themselves, and the lack of
homogeneity of cell contents, afford insurmountable difficulties to this
conception as a basis for the study of cell activities. The other, and
seemingly more reasonable, conception is that these bodies exist in the
form of colloidal complexes, whose composition may vary within wide limits
and whose reactions are responsive to the usual phenomena incident to the
colloidal condition of matter.