It is obvious that the compound thus formed still contains a free NH2group 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, asmany 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 enzymepepsin.
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 lettersa,b,c, andd, the arrangement may be in the ordersabcd,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 NH2groups, 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.
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:
Monoamino-monocarboxylic acids:Glycine, C2H5NO2, CH2NH2·COOH, amino-acetic acid.Alanine, C3H7NO2, CH3·CHNH2·COOH, amino-propionic acid.Serine, C3H7NO3, CH2OH·CHNH2·COOH, oxy-amino-propionic acid.Valine, C5H11NO2,, amino-isovalerianic acid.Leucine, C6H13NO2,, amino-isocaproic acid.Isoleucine, C6H13NO2,, amino-methylethyl-propionic acid.Phenylalanine, C9H11NO2,, phenyl-amino-propionic acid.Tyrosine, C9H11NO3,, paraoxy-phenylalanine.Cystine, C6H12N2O4S2, HOOC·CHNH2·CH2S—SH2C·CHNH2·COOH, di(thio-amino-propionic acid).Monoamino-dicarboxylic acids:Aspartic acid, C4H7NO4, HOOC·CH2·CHNH2·COOH, mino-succinic acid.Glutamic acid, C5H9NO4, HOOC·CH2·CH2·CHNH2·COOH, amino-glutaric acid.Diamino-monocarboxylic acids:Ornithine, C5H12N2O2, H2N·CH2·CH2·CH2·CHNH2·COOH, di-amino-valerianic acid.Lysine, C6H14N2O2, H2N·CH2·CH2·CH2·CH2·CHNH2·COOH, di-amino-caproic acid.Arginine, C6H14N4O2,, guanidine-amino-valerianic acid.Di-amino-oxysebacic acid, C11H12N2O3.Di-amino-trioxydodecanic acid, C12H26N2O3.Monoimido-monocarboxylic acids:Proline, C5H9NO2,, pyrrolidine-carboxylic acid.Oxyproline, C5H9NO3, proline with one (OH) group.Monoimido-monoamino-monocarboxylicacids:Histidine, C6H9N3O2,, imidazole-amino-propionic acid.Tryptophane, C11H12N2O2,, indole-amino-propionic acid.
As has been said, other amino-acids are being found, from time to time, as additional proteins are examined, or as better methods of examination of the cleavage products of the natural proteins are devised.
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:
Gliadin(whaet).Hordein(barley).Zein(corn).Legumin(vetch).Edestin(hemp).Globulin(squash seed).Amandin(almonds).71.4678.1685.2762.6582.3855.7759.00Glycine0.020.000.000.393.800.570.51Alanine2.000.439.791.153.601.921.40Valine0.210.131.881.366.200.260.16Leicine5.615.6719.558.8014.507.324.45Proline7.0613.739.044.044.102.822.44Phenylalanine2.355.036.552.873.093.322.53Aspartic acid0.58.....1.713.214.503.305.42Glutamic acid42.9843.1926.1718.3018.8412.3523.14Serine0.13?1.02?0.33??Cystine0.45???1.000.23?Tyrosine1.201.673.552.422.133.071.12Arginine3.162.161.5511.0614.1714.4411.85Histidine0.611.280.432.942.192.631.58Lysine...............3.991.651.990.70TryptophanepresentpresentpresentpresentpresentpresentpresentAmmonia5.114.873.642.122.281.553.70
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 thedifferent physiological functions which the proteins have to perform in the animal body: thus,tryptophaneis 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; whilearginineseems to be definitely associated with the reproductive function; andcystine, 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.
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 allcolloidalin 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 becoagulated, forming a semi-solidgel, 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 NH2groups 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 containstyrosine, or atyrosine-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 ontyrosine. 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 (orbetter, 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 reactionfor furfural will be shown by those proteins which contain a carbohydrate group. In applying this test, the solution to be tested is first treated with a few drops of an alcoholic solution of α-naphthol, and then concentrated sulfuric acid is poured carefully down the sides of the test-tube. If carbohydrates are present, either free or as a part of a protein molecule, a red-violet ring forms at the juncture of the two liquids.
(f)Sulfur Test.—If a drop of a solution of lead acetate be added to a solution containing a protein, followed by sufficient sodium hydroxide solution to dissolve the precipitate which forms, and the mixture is heated to boiling, a black or brown coloration will be produced if the protein contains cystine, the sulfur-containing amino-acid.
FOOTNOTES:[5]Since the proteins are essentiallycolloidalin 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 toChapter XVfor an explanation of any terms used here, which may not be familiar to him; and that after the study ofChapter XV, he return to this chapter dealing with the proteins for an illustrative study of the applications of the principles presented there.
[5]Since the proteins are essentiallycolloidalin 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 toChapter XVfor an explanation of any terms used here, which may not be familiar to him; and that after the study ofChapter XV, he return to this chapter dealing with the proteins for an illustrative study of the applications of the principles presented there.
[5]Since the proteins are essentiallycolloidalin 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 toChapter XVfor an explanation of any terms used here, which may not be familiar to him; and that after the study ofChapter XV, he return to this chapter dealing with the proteins for an illustrative study of the applications of the principles presented there.
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:
Simple proteins, which yield only amino-acids when hydrolyzed.Conjugated proteins, compounds of proteins with some other non-protein group.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:
The Simple ProteinsAlbumins—soluble in water and dilute salt solutions, coagulated by heat.Globulins—insoluble in water, soluble in dilute salt solutions, coagulated by heat.Glutelins—insoluble in water or dilute salt solutions, soluble in dilute acids or alkalies, coagulated by heat.Prolamins—insoluble in water, etc., soluble in 80 per cent alcohol.Histones—soluble in water, insoluble in ammonia, not coagulated by heat.Protamines—soluble in water and ammonia, not coagulated by heat, yielding large proportions of diamino-acids on hydrolysis.Albuminoids—insoluble in water, salt solutions, acids, oralkalies.Conjugated ProteinsChromoproteins—compounds of proteins with pigments.Glucoproteins—compounds of proteins with carbohydrates.Phosphoproteins—proteins of the cytoplasm, containing phosphoric acid.Nucleoproteins—proteins of the nucleus, containing nucleic acids.Lecithoproteins—compounds of proteins with phospholipins.Lipoproteins—compounds of proteins with fats, existence in nature doubtful, artificial forms easily prepared.Derived ProteinsPrimary protein derivatives.Proteans—first products of hydrolysis, insoluble in water.Metaproteins—result from further action of acids or alkalies, soluble in weak acids and alkalies, but insoluble in dilute salt solutions.Coagulated proteins—insoluble forms produced by the action of heat or alcohol.Secondary protein derivatives.Proteoses—products of hydrolysis, soluble in water, not coagulated by heat, precipitated by saturation of solution with ammonium sulfate.Peptones—products of further hydrolysis soluble in water, not coagulated by heat, not precipitated by ammonium sulfate, give biuret reaction.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:
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, otherthan 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.
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, ortryptophane. It is not sufficient to support animal life and promote growth, if used as an exclusive source for protein for food.
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 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, andvice versa, under the action of the corresponding carbohydrate-attacking enzymes.
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.
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,Monographson 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,Monographson 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,Monographson Biochemistry, London, 1909.Underhill, F. P.—"The Physiology of the Amino-acids," 169 pages, 13 figs. 1 plate. Yale University Press, 1915.
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,Monographson 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,Monographson 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,Monographson Biochemistry, London, 1909.
Underhill, F. P.—"The Physiology of the Amino-acids," 169 pages, 13 figs. 1 plate. Yale University Press, 1915.
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 individualmolecules, but a considerable proportion of these molecules separate into their constituentions. 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 whencarriedon 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 livingorganism ("in vivo"), is due to the universal presence in the living protoplasm of certain organic catalysts, known asenzymes.
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, andin 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.
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 simplehydrolyses, 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 asmaltase(which acts on all α-glucosides) and emulsin (which acts on all β-glucosides) which act catalytically upon groups of considerable numbers of similar compounds.
Enzymes, like all other catalysts, act more energetically at increased temperatures; but for each particular enzyme there is an "optimum temperature," (usually between 40° and 65°) above which the destructive effect of the temperature upon the enzyme itself more than offsets the accelerating influence of the increased temperature. At still higher temperatures (usually 80° to 100°) the enzymes are "killed," i.e., rendered permanently inactive. All enzymes are "killed" by boiling the solutions in which they are contained. Dry preparations of enzyme material can withstand somewhat higher temperatures, for somewhat longer periods of time, than can the same enzyme in moist condition or in solution.When an enzyme has once been inactivated by heating, or "killed," it can never be restored to activity again.
Enzymes are extremely sensitive to acids, bases, or salts, their activity being often enormously enhanced or, in other cases, entirely inhibited, by the presence in the reacting medium of very small amounts of free acids, or bases, or even of certain neutral salts. For example, pepsin, the enzyme of the stomach will act only in the presence of a slightly acid medium and is wholly inactive in a mixture which contains even the slightest amount of free alkaline material; while trypsin, the similar enzyme of the intestine, acts only under alkaline conditions. Practically all enzymes are rendered inactive, but not destroyed, by the presence of either acid or alkali in excess of N/10 strength. Many will act only in the presence of small quantities of certain specific neutral salts; while, on the other hand, other salts are powerful inhibitors of enzyme action. Enzymes often differ from the protoplasm which secretes them in their response to antiseptics, such as toluene, xylene, etc., which inhibit the activity or growth of the cell, but have no effect upon the activity of the enzymes which it contains.
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, theymay 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 followingChapter).
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 suffixin, 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 suffixase. Thus, cellulase is the enzyme which accelerates thehydrolysisof 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 thesubstrate.
Most enzymes are catalysts for hydrolysis reactions and are, hence, classed ashydrolyticin 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 CO2from COOH groups are "carboxylases," etc.
The hydrolytic enzymes are furthersubdividedinto the sucroclastic (sugar-splitting), or sucrases; the lipoclastic (fat-splitting), or lipases; the esterases (ester-splitting); proteoclastic (protein-splitting), or proteases; etc.
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 thetimerequired to carry a unit quantity of the substrate material through any determined stage of chemical change, or thequantityof 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.