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