CHAPTER XIII
PROTEINS
The proteins are the most important group of organic components of plants. They constitute the active material of protoplasm, in which all of the chemical changes which go to make up the vital phenomena take place. Combined with the nucleic acids, they comprise the nucleus of the cell, which is the seat of the power of cell-division and, hence, of the growth of the organism. Germ-cells are composed almost exclusively of protein material. Hence, it is not an over-statement to say that proteins furnish the material in which the vital powers of growth and repair and of reproduction are located. A recognition of their importance is reflected in the use of the name "protein," which comes from a Greek word meaning "pre-eminence," or "of first importance."
In addition to the proteins which constitute the active protoplasm, plants also contain large amounts of reserve, or stored, proteins, especially in the seeds. In the early stages of growth, the proteins are present in largest proportions in the vegetative portions of the plant; but as maturity approaches, a considerable proportion of the protein material is transferred to the seeds.
GENERAL COMPOSITION OF PROTEINS
The plant proteins are fairly uniform in their percentage composition. The analyses of some sixteen different plant proteins show the following maximum limits of percentages of the different chemical elements which they contain: Carbon, 50.72-54.29; hydrogen, 6.80-7.03; nitrogen, 15.84-19.03; oxygen, 20.86-24.29; sulfur, 0.17-1.09. Animal proteins vary more widely, both in percentage composition and in properties, than do those of plant origin.
Protein molecules are very large and, in the case of the so-called "conjugated proteins" in particular, their structure is very complex. The molecular weight of some of the proteins has been determined directly, in the case of those particular ones which can be prepared in proper form for the usual determination of molecular weight by the osmotic pressure method; and has been computed for various others, from the percentage of sulfur found on analysis, or (in the case of the hæmoglobin of the blood) from the proportion by weight of oxygen absorbed. From these determinations and computations, the following formulas for certain typical proteins have been calculated: for zein (from Indian corn), C_{736}H_{1161}N_{184}O_{208}S_{3}; for gliadin (from wheat), C_{685}H_{1068}N_{196}O_{211}S_{5}; for casein (from milk), C_{708}H_{1130}N_{180}O_{224}S_{4}P_{4}; for egg-albumin, C_{696}H_{1125}N_{175}O_{220}S_{8}. These few examples will serve to illustrate the enormous size and complexity of the protein molecule. The conjugated proteins are still more complex than the simple proteins whose formulas are here presented.
Fortunately for the purposes of the study of the chemistry of the proteins, however, it has been found that most of the common plant proteins, known as the "simple proteins," can easily be hydrolyzed into their constituent unit groups, which are the comparatively simple amino-acids, whose composition and properties are well understood. A study of the results of the hydrolysis of some twenty common plant proteins has shown that it is rarely possible to recover the amino-acids in sufficient quantities to account for a full 100 per cent of the material used, the actual percentage of amino-acids recovered usually totaling from 60 to 80 per cent. The remaining material is supposed to be also composed of amino-acids which are linked together in some arrangement which is not broken apart by any method of hydrolysis which has yet been devised. This view is borne out by the fact that substances which exhibit all the characteristic properties of proteins have been artificially synthetized, by using only amino-acid compounds. Animal proteins often show a much larger proportion of unhydrolyzable material than do plant proteins.
AMINO-ACIDS AND PEPTID UNITS
The products of hydrolysis of the common simple proteins are all amino-acids. These are ordinary organic acids with one (or more) of the hydrogen atoms of the alkyl group replaced by a --NH_{2} (or sometimes by a --NH--) group. They may be regarded as ammonia, NH_{3}, with one of its hydrogen atoms replaced by an acid radical; or as the acid with one of its hydrogens replaced by the NH_{2} group. For example, an amino-acid derived from acetic acid, CH_{3}·COOH, is glycine, or amino-acetic acid, CH_{2}NH_{2}·COOH; from propionic acid, CH_{3}·CH_{2}·COOH, there may be obtained either [alpha]-amino-propionic acid, CH_{3}·CHNH_{2}·COOH, or [beta]-amino-propionic acid, CH_{2}NH_{2}·CH_{2}·COOH, etc.
All of the amino-acids which result from the hydrolysis of proteins are [alpha]-amino-acids, that is to say, the NH_{2} group is attached to the [alpha]-carbon atom, i.e., the one nearest to the COOH group. Hence, the general formula for all the amino-acids which are found in plants is R·CHNH_{2}·COOH.
These amino-acids contain both the basic NH_{2} group and the acid COOH group. For this reason, they very easily unite together, in the same way that all acids and bases unite, to form larger molecules, the linkage taking place between the basic 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.