Class and Type.Enzyme.Substrate.End-products.Found in.I. Hydrolases(a) EsterasesLipaseFatsGlycerol and fatty acidsOily seeds(b) CarbohydrasesSucrase or invertaseSucroseGlucose and fructoseYeastsMaltaseMaltose and all α-glucosidesGlucose, etc.Barley maltDextrinaseDextrinMaltoseMaltInulaseInulinFructoseArtichokes, etc.Amylase or diastaseStarchMaltoseMalt etc.CellulaseCelluloseMaltoseBacteriaPectinasePectoseArabinoseFruitsCytaseHemi-cellulosesMono-saccharidesNuts, seeds, etc.(c) GlucosidasesEmulsinAmygdalin and all β-glucosidesGlucose, etc.Almond kernels, etc.Maltaseα-glucosidesGlucose, etc.Barley maltMyrosinSulfur-containing glucosidesGlucose, etc.Mustard seedsRhamnaseXanthorhamninRhamnose, etc.Rhamnus spp.PhytasePhytinInosite and H3PO4Bran coats of seeds(d) ProteasesErepsinProteinsAmino-acidsMany plantsPapainProteinAmino-acidsPapawsBromelinProteinAmino-acidsMany plantsNucleaseNucleo-proteinsProteins and nucleic acidMany plantsII. Oxidases(a) Catalases........Hydrogen peroxideWater and oxygenNearly all plants(b) Peroxidases........Organic peroxides"Active" oxygenNearly all plants(c) Oxidases........ChromogensAlcohols and phenolsPigmentsAcidsMany plantsMany plants(d) Reductases........................Many plantsIII. DeaminasesUreaseUreaAmmonia and CO2GuanaseGuanineXanthineAdenaseAdenineHypoxanthineIV. Carboxylases(a)........Keto-acidsAldehydes and CO2(b)........Amino-acidsAmines and CO2V. Coagulation enzymesPectaseCoagulates pectic bodies........FruitsVI. Fermentation enzymesZymaseGlucose, etc.Alcohol and CO2YeastsLactic acid fermentFatty acidsLactic acidBacteriaButyricacid fermentFatty acidsButyric acidBacteria
The above list includes only the more common and best-known plant enzymes. It seems reasonable to suppose that for every individual type of organic compound which may occur in general plant groups, or even in single species, there is a corresponding enzyme available to affect its physiological alterations. Indeed, new preparations of active enzymes from special types of plants and new evidences of the existence of enzymes in various plant organisms are continuously being reported.
A few of the most common specific representatives of individual groups of enzymes may be briefly described, as follows:
Amylase(ordiastase, as it was first named and is still commonly called) is probably the most widely distributed enzyme of plants. It is found in practically all bacteria and fungi; in practically all seeds (it has been found in active form in seeds which were known to be over fifty years old); in all roots and tubers; and in practically all leaves, where it is located in the stroma of the chloroplasts.
It appears to exist in two modifications, known, respectively, as (a) translocation diastase and (b) diastase of secretion. The first form is found in the cells of ungerminated seeds, in leaves, shoots, etc. It remains in the cells where reserve starch is stored and aids in the transformation of starch into soluble materials for translocation from cell to cell. It is active at a lower temperature than the second form, its optimum temperature being 45° to 50°. The second form is secreted by the scutellum, and perhaps by the aleurone cells, of germinating seeds, being produced by special glandular tissue. It aids in the hydrolysis of the starch for the use of the growing embryo. Its optimum temperature is 50° to 55°.
The activity of amylase is accelerated by the presence of small quantities of neutral salts, especially by sodium chloride and disodium phosphate. It acts best in neutral solutions, its activity being inhibited, although the enzyme itself is not destroyed, by the presence of more than minute traces of free mineral acid or alkali.
Sucrase(or invertase) is present in almost all species of yeasts, where it serves to convert unfermentable sucrose into glucose and fructose, which are readily fermentable. Invertase is also present in moulds and othermicroorganisms; and in the buds, leaves, flowers, and rootlets of those higher order plants which store their carbohydrate reserves in the form of sucrose. It appears that sucrose, while easily soluble, is not readily translocated, or utilized,by plants until after it has been hydrolyzed into its constituent hexoses.
The optimum temperature for invertase is 50° to 54°; it is killed if heated, in the moist condition, to 70°. Its activity is increased by the presence of small amounts of free acids; but is inhibited by free alkalies.
Zymaseis the active alcoholic fermentation enzyme of yeasts. It accelerates the well-known reaction for the conversion of hexose sugars into alcohol and carbondioxide, namely,
C6H12O6= 2C2H5OH + 2CO2.
Because of its scientific interest and industrial importance in the fermentation industries, its action has been extensively studied. It acts only in the presence of soluble phosphates and of a coenzyme (see below) which is dialyzable and not destroyed, which is probably an organic ester of phosphoric acid. The significance of the molecular configuration of the hexose sugars in their susceptibility to action by zymase has already been discussed in detail (seepage 56).
The optimum temperature for zymase action is 28° to 30°. The enzyme is killed by heating to 45° to 50° in solution, or to 85° if in dry preparation.
Proteasesof the erepsin type, i.e., those which break proteins down to amino-acids instead of only to the proteose or peptone stage, as is characteristic of the enzymes of the trypsin type, are widely distributed in plants. Except in the case of the two which occur in large amounts in certain special fruits (papain in papaws, and bromelin in pineapples), they are very difficult to prepare in pure form for study. In general, all proteolytic actions, even when accelerated by active enzymes, proceed much more slowly than do the hydrolyses of carbohydrates or fats. It seems that metabolic changes of the complex protein molecules are much more difficult to bring about and take place much more slowly than do those of the energy-producing types of compounds.
The presence of proteolytic enzymes in most vegetative cells, and in seeds, may be demonstrated, however, by studying the action of extracts of these tissues upon soluble proteins. The best-known example of this type of enzymes is the protease of yeast; but similar ones may be found in germinating seeds. Thesevegetable proteases are usually most active in neutral or only faintly alkaline solutions, and their activity is nearly always inhibited by even traces of free acids.
Most laboratory studies of proteolytic enzymes are carried on with preparations of the powerful members of this class of enzymes which are found in the digestive tract of animals, namely, the pepsin of the gastric juice, which acts in the acid medium, in the stomach, and the trypsin of the pancreatic juice, which acts in the alkaline medium of the intestinal tract. But even these powerful proteases require several hours for the transformation of an amount of soluble albumin into its amino-acid constituents which is equivalent to the amount of starch which is hydrolyzed to maltose by diastase in a very few minutes.
Enzymes which govern oxidative changes, known respectively, ascatalasesandoxidases, are almost universally present in plants. Catalase decomposes peroxides, with the liberation of free oxygen. It is, therefore, necessary to the final step in the process of photosynthesis, as elucidated by Usher and Priestley (seepage 26), and serves to prevent the destructive action of hydrogen peroxide upon chlorophyll. The almost universal presence of oxidases in plant tissues has been repeatedly demonstrated. They are present in especially large amounts in tissues which are being acted upon by parasitic fungi or are combating unfavorable conditions of growth. The oxidases, in such cases, seem to be the agents by which the plant is able to stimulate its metabolic activities to overcome the unfavorable environment for its normal development.
In vegetables and fruits, the common browning, or blackening, of the tissues when cut surfaces are exposed to the air has been demonstrated to be due to the catalytic oxidation of the tannins or of certain amino-acids, especiallytyrosine, under the influence of the oxidases which are present in the tissues. In fact, most pigmentation phenomena are due to changes in the oxygen content of the chromogens of the cells of the plant, under the influence of the oxidases which are present in the protoplasm of the cells in question. Hence, the oxidases may be said to be the controlling agencies for both the energy-absorbing activities and for respiration in plants.
The mechanism by which an enzyme accomplishes its catalytic effects has been the object of extensive studies during recent years, especially since the discovery by Büchner that enzymes could be isolated in solutions entirely free from the disturbing influence of growing cells. Several theories concerning the mode of this catalytic action have been advanced. The earliest and simplest of these was that the enzyme simply creates an environment favorable for the particular chemical reaction to take place, as by exposing large surfaces of the substance in question to the action of the hydrolytic, or other effective, agent, by means of surface adsorption of the substrate material on the colloidal enzyme.
However, more recent investigations clearly indicate that there is an actual combination between the substrate material and the enzyme, which combination then breaks down with a resultant change in the substrate material and a freeing of the enzyme for repeated recombination with additional substrate, with the net result that the chemical change in the substrate material is enormously accelerated. That such a combination between substrate and enzyme actually exists has been demonstrated in two different ways: (a) experimentally, by mixing together solutions of an enzyme and of its substrate, each of which is filterable through paper or through a porous clay filter, with the result that the active material in the combined solutions will not pass through these same filters; and (b) mathematically, by a study of the curves representing the reaction velocities of typical reactions which are proceeding under the influence of an enzyme, which show that so long as there is a large excess of substrate material present, the accelerating influence of the catalyst is uniform over given successive periods of time, but that when the quantity of substrate material becomes smaller than that which permits the maximum combining power of the enzyme to be exercised, the reaction velocity immediately slows up.
Again, the fact that the specificity of the action of an enzyme, i.e., the limitation of the action of that enzyme to a specific single compound or group of similar compounds, is definitely related to the molecular configuration of the molecule of the substrate, as has been found to be true in all those cases where the molecular configuration of the substrate material has been established (seepages 56 to 58), is an added indication that there is some kind of a union between the enzyme and the substrate as a first step in the catalytic process.
As to the nature of this supposed combination of substrate and enzyme, two theories are held. The first is that this union is in the form of an actual molecular combination, or chemical compound, and the other is that it is a purely physical, or colloidal complex. The latter view has by far the greater weight of theoretical and experimental evidence in its support. The relation of electrolytes to the catalytic effect of enzymes, the appearance of the reacting masses under theultramicroscope, and the effect of heat upon the reacting mixtures, all point to the conclusion that the phenomenon is colloidal rather than molecular in character. This view also makes the remarkable catalytic effects which take place in living protoplasm, which undoubtedly exists in the colloidal condition, much more easily understood. This phase of the matter will be much more apparent after the chapter dealing with the physical chemistry of the protoplasm has been studied.
A further indication that the mechanism of enzyme activity is colloidal in character lies in the fact that, so far as is known, all reactions which are catalyzed by specific enzymes are reversible and the same enzyme will accelerate the velocity of the reaction in either direction, the direction in which the reaction goes being determined by the conditions surrounding the reacting material at the time. It was formerly supposed that enzymes catalyze only decomposition reactions and that the synthetic reactions of living tissues are produced by means of some other force or agency. This view supported the idea of a chemical union of the enzyme with the substrate which, when it breaks down, breaks the molecule of the substrate material into some simpler form, or forms. But it is now known that the reaction which is influenced by the enzyme will be catalyzed in either direction by the specific enzyme which "fits" the particular substrate material at every point of its molecular configuration, as the glove fits the hand. The contrast between this fitting of the enzyme to the entire configuration of the molecule, and the union at a single point or group which is characteristic of chemical linkages, is apparent. As examples of the synthetic action of the same enzyme which, under other conditions, accelerates the decomposition of the same material, there may be cited the demonstrated synthesis of isomaltose from glucose by maltase; the production of ethyl butyrate from alcohol and butyric acid; and the synthetic production of artificial fats, by the aid of the pancreatic lipase; and the apparent synthesis of a protein from the same amino-acids which may be obtained from it by hydrolysis under the influence of the same protease, but under different environmental conditions.
The activity of enzymes is strongly influenced by the presence in the solution of other bodies, usually, although not always, electrolytes. This is probably due, in most cases at least, to the action of the electrolyte upon the colloidal condition of the enzyme. All enzymes do not respond alike to the action of the same electrolyte, however. The activity of certain enzymes is enormously increased by the presence of a small amount of acid; while the action of another may be absolutely inhibited by the same acid in the same concentration. Thus, the activity of the amylase found in the endosperm of many seeds is instantly stopped by adding to the solution enough sulfuric acid to make it two-hundredth normal in strength; while the same concentration of acid actually accelerates the activity of some of the proteases.
Formaldehyde, hydrocyanic acid, and soluble fluorides usually inhibit both the activity of a cell and of the enzymes which it contains; while other antiseptics, such as toluene, xylene, etc., prevent the growth of the cell, or organism, without interfering with the activity of the enzymes which may be present. By the use of this latter type of antiseptics, it is possible to distinguish between chemical changes which are involved in the actual development of a cell and those which can be brought about in other media by means of the enzymes which are contained in the cell.
Any substance which increases the catalytic activity of an enzyme is known as an "accelerator," or "activator"; while one which prevents this activity is called an "inhibitor," or "paralyzer."
A type of accelerating influence quite different from that of electrolytes is found in the effect of certain amino-acids upon enzyme action. The influence of small amounts of asparagine in enormously increasing the hydrolytic effect of amylase is an example. There is no known explanation for this type of activation of the enzyme.
The influence of activators, or inhibitors, in providing favorable or unfavorable conditions for the action of an enzyme, should not be confused with the relation to the enzyme itself of what are known as "coenzymes" and "antienzymes," discussed in the following paragraph.
In the cases of many enzymes of animal tissues, it has been found that they are absolutely inactive unless accompanied by some other substance which is normally present in the gland, or protoplasm, which secretes them. Thus, the bile salts are absolutely necessary to the activity of trypsin, in its characteristic protein-splitting action. Such substances are known as "coenzymes." They can usually be separated from their corresponding enzymes by dialysis, the coenzyme passing through the parchment membrane. Such coenzymes are not killed by boiling the dialyzate, and the activity of the enzyme is restored by adding the boiled dialyzate to the liquid which remains within the dialyzer.
The best known example of a coenzyme in plant tissues is in connection with the activity of the zymase of yeast cells. If yeast juice be filtered through a gelatin filter, the colloidal enzymes which are left behind are entirely inactive in producing fermentation, but may be restored to activity again by mixing with the filtrate. An examination of this filtrate, which contains the coenzyme for zymase, shows that it contains soluble phosphates and some other substance whose exact nature has not yet been determined, both of which are necessary to the activity of the zymase. The phosphates seem to enter into some definite chemical combination with the substrate sugars, while the other coenzyme seems to be necessary in order to make possible the final breaking down of the sugar-phosphate complex by the zymase. This phenomenon of coenzyme relationship is not very frequently observed in plant enzyme studies, probably because the coenzyme (if there be such, in the case which is under observation) usually accompanies the enzyme itself through the various processes of extraction and purification of the material for study. However, care must betaken in all cases when dialysis is employed, to see that a possible coenzyme is not separated from an otherwise active preparation.
An entirely different type of phenomenon is that exhibited by "antienzymes." These are found in the various intestinal worms which live in the digestive tracts of animals; and prevent the digestive action of the enzymes of the stomach and intestines upon these worms. Probably similar "antienzymes" are located in the mucous linings of the intestinal tract itself, and serve to prevent the auto-digestion of these organs by the active enzymes with which they are almost continually in contact.
The difference between an antienzyme, which protects material which would otherwise be subject to the attack of an enzyme, and an inhibitor, which renders the enzyme itself inactive, is apparent.
So far as is known, however, no such substances as antienzymes are present in plant tissues; although the question as to why the proteoclastic enzymes which are elaborated by a given mass of protoplasm do not attack the protoplasm itself, might well be raised.
It is apparent that, since enzymes are produced by protoplasm for the special needs of any given moment or stage of development, there must be a preliminary stage, or condition, in which they do not exert their characteristic catalytic effect. When in this stage, the compound is known as "proenzyme," or "zymogen." In this stage, it is inactive, but can be made to exhibit its catalytic effect, usually by bringing it into contact with a suitable activator. When once so activated, however, it cannot be returned again to the inactive state.
This phenomenon has been studied in connection with the zymogens of the digestive proteases, pepsin and trypsin. Trypsinogen may be rendered active by contact with either calcium salts or with another substance (apparently itself an enzyme) known as enterokinase, which is secreted in the intestinal tract.
Similarly, proenzymes have been reported as occurring in numerous plant tissues. These proenzymes are believed to be present in the plant cells in the form of definite characteristic granules, which may be observed under the microscope, and which disappear when the enzyme becomes active. Thus, "proinulase" has been reported as occurring in artichoke tubers: "prolipase," in castor beans; "proinvertase," in several species of fungi; and, probably, "prooxidase," in tobacco leaves. In the case of the last-named zymogen, it has been observed that after the zymogen has been once activated, as in response to the need for increased activity due to the entrance of the germs of certain leaf-diseases, it can once again produce a second supply of the enzyme, but the process cannot again be repeated.
Calcium salts, or very dilute acids, are usually energetic activators of proenzymes.
There can be no doubt that enzymes exert a tremendously important influence in vital phenomena, by determining the rate at which the chemical changes which are involved in these phenomena shall proceed. Since they do not initiate reactions, and since they may catalyze reversible reactions in either direction, it cannot be said that they determine the type of reactions which will take place in any given mass of protoplasm; but, undoubtedly, they do exert a determining influence upon the rate at which the reaction will proceed, after the protoplasmic activity has determined the direction in which it shall go.
Without the intervention of these catalyzing agents, it would be impossible for reactions between these non-ionized organic components of the cell contents to come to completion with anything like the marvelous rapidity with which these changes must take place in order to permit the organism to grow, to perform its necessary vital functions, or to adjust itself to the changes in its environmental conditions.
Since the number of different reactions which take place within a living cell is very great, and since these chemical changes are extremely variable in type, it follows that the number of different enzymes which must exist in either a plant or an animal organism is likewise very large. For example, fourteen different enzymes have been isolated from the digestive system, and at least sixteen from the liver, of animals. They are universally present in living protoplasm of every kind, from the most minute bacterium to the largest forest trees, in the plant kingdom; and from the amœba to the whale, in animals.
While there is a great variety of enzymes which may be produced by a single individual organism, the same enzyme may be found in the greatest variety of organisms; as, for example, the protease trypsin, which has been found in several species of bacteria, in the carnivorous plant known as "Venus' Fly Trap," and in the human pancreas, as well as that of all other animals.
From the discussions which have been presented in this chapter, it is apparent that the enzymes play a tremendously important part in vital phenomena, by controlling the rate at which the biochemical reactions take place in the cells of the living organism.
The means by which the protoplasm elaborates these all-important chemical compounds are as yet absolutely unknown. Even the nature of the enzymes themselves is still a matter of speculation and study. Much intensive study is needed and should be given to these matters, for the purpose of elucidating the methods by which the enzymes accomplish their remarkable catalytic effects, and, if possible, the actual chemical nature of the enzymes themselves. It is conceivable, of course, that if the latter object of these studies should ever be reached, it might be possible to synthetize enzymes artificially, and so to develop a means for the artificial duplication of the synthesis of organic compounds with the same velocity that this is done in the plant cells. Such a result would have a scientific interest fully as great as did Wöhler's artificial synthesis of urea, which proved that there is no essential difference in character between the compounds which are the products of living organisms and those which are produced in the laboratory; and, at the same time, might have an immensely more important practical bearing, since it would lead the way to the artificial production of the carbohydrates, proteins, fats, etc., for which we are now dependent upon plant growth as the source of these materials for use as human food.
Bayliss, W. M.—"The Nature of Enzyme Action," 186 pages,Monographson Biochemistry, London, 1919 (4th ed.).Euler, H., trans. byPope, T. H.—"General Chemistry of the Enzymes," 319 pages, 7 figs., New York, 1912.Effront,J.,trans. byPrescott, S. C.—"Enzymes and their Application,—Enzymes of the Carbohydrates," 335 pages, New York, 1902.Effront, J., trans. byPrescott, S. C.—"Biochemical Catalysts in Life and Industry—Proteolytic Enzymes," 763 pages, New York, 1917.Green, J. R.—"The Soluble Ferments and Fermentation," 512 pages, Cambridge, 1901, (2d ed.).Grus, J.—"Biologie und Kapillaranalyse der Enzyme," 227 pages, 58 figs., 3 plates, Berlin, 1912.Harden, A.—"Alcoholic Fermentation," 156 pages, 8 figs., Monographs on Biochemistry, London, 1914.Plimmer, R. H. A.—"The Chemical Changes and Products Resulting from Fermentations," 184 pages, London, 1903.Oppenheimer, C., trans. byMitchell, C. A.—"Ferments and their Actions," 343 pages, London, 1901.
Bayliss, W. M.—"The Nature of Enzyme Action," 186 pages,Monographson Biochemistry, London, 1919 (4th ed.).
Euler, H., trans. byPope, T. H.—"General Chemistry of the Enzymes," 319 pages, 7 figs., New York, 1912.
Effront,J.,trans. byPrescott, S. C.—"Enzymes and their Application,—Enzymes of the Carbohydrates," 335 pages, New York, 1902.
Effront, J., trans. byPrescott, S. C.—"Biochemical Catalysts in Life and Industry—Proteolytic Enzymes," 763 pages, New York, 1917.
Green, J. R.—"The Soluble Ferments and Fermentation," 512 pages, Cambridge, 1901, (2d ed.).
Grus, J.—"Biologie und Kapillaranalyse der Enzyme," 227 pages, 58 figs., 3 plates, Berlin, 1912.
Harden, A.—"Alcoholic Fermentation," 156 pages, 8 figs., Monographs on Biochemistry, London, 1914.
Plimmer, R. H. A.—"The Chemical Changes and Products Resulting from Fermentations," 184 pages, London, 1903.
Oppenheimer, C., trans. byMitchell, C. A.—"Ferments and their Actions," 343 pages, London, 1901.
Reference has frequently been made, in preceding chapters, to the fact that proteins, enzymes, lipoids, etc., exist in the protoplasm of plants and animals in the colloidal condition. The properties and uses of these compounds by plants depend so much upon this fact that, before proceeding to the consideration of the actual physical chemistry of protoplasm itself, it will be appropriate and profitable to give some attention to the nature and significance of the colloidal condition of matter and of some of the phenomena which grow out of it.
Every discussion of the colloidal condition in general properly begins with reference to the work of the English physicist, Thomas Graham, who carried on his investigations of the so-called "colloids" through a period of forty years, beginning with 1851. His most important results were published, however, from 1861 to 1864. Graham studied the diffusibility of substances in solution through the parchment membrane of a simple dialyzer. As a result of his earlier investigations, he divided all the chemical compounds which were known to him into two groups, which he called "crystalloids" and "colloids," respectively, the first including those substances which readily diffused through the parchment membrane and the second those which diffused only very slowly or not at all. He at first thought that crystalloids are always inorganic compounds, while colloids are of organic origin. He soon learned, however, that this distinction in behavior is not always related to the organic or inorganic nature of the compound. He further discovered that the same individual chemical element or compound may exist sometimes in crystalloidal, and sometimes in colloidal, form. This latter discovery led to the conclusion that diffusibility depends upon thecondition, rather than upon thenature, of the material under observation.
As a result of the long series of investigations which were stimulated by Graham's work, the modern conception is that diffusibility is aconditionof matter when in minute subdivision, or in solution, in some liquid, as contrasted with itsstate, or condition, when existing alone. That is, thestateof a substance may be either gaseous, liquid, or solid; and itsconditionwhen in solution may be either crystalloidal or colloidal. Substances which are in crystalloidal form, in true solution, exist there in molecular or ionized condition; but, as will be pointed out below, when in the colloidal condition they exist in aggregates which are somewhat larger than molecules, but not large enough to be visible as individual particles under the ordinary microscope, even under the highest magnification which has yet been obtained. Colloidal particles are, however, generally visible under the Zigmondy "ultramicroscope." (See below.)
The use of the word "colloid" as a noun, or as the name for a substance which is in the colloidal condition, is of the same nature as the use of the words "gas," "liquid," and "solid," in such statements as "ice is a solid," "water is a liquid," or "steam is a gas," etc.; i.e., the noun represents a state or condition rather than an actual object or thing. Hence, the expression "enzymes are colloids," means only that enzymes exist in the colloidal condition, and not that enzymes represent a definite type of substances having the group name "colloids."
When one substance is distributed through the mass of another substance, the mixture is said to be a "two-phase system," composed of thedispersed phase, or substance, and thedispersion medium, orcontinuousphase, through which the other substance is distributed. The following examples illustrate the possibilities of such two-phase systems:
Dispersion medium a gas.Disperse phase a liquid—mist in the air.Disperse phase a solid—smoke or dust in air.Dispersion medium a liquid.Disperse phase a gas—foams.Disperse phase a liquid—emulsions.Disperse phase a solid—suspensions.Dispersion medium a solid.Disperse phase a gas—solid foams, pumice stone, etc.Disperse phase a liquid—liquid inclusions in minerals.Disperse phase a solid—alloys, colored glass, etc.
Although the same general principles of physical chemistry apply to all two-phase systems, the term "colloidal condition" is commonly used only in connection with a particular type of dispersions, in which the dispersion medium is a liquid and the dispersed material is either a solid or a liquid.
Thorough and careful studies have shown that when a solid or a liquid is introduced into another liquid, and becomes dispersed or distributed through it, the mixture may be either a true solution, a colloidal solution, or a mechanical suspension. The characteristic differences between these three conditions may be tabulated as follows: although the significance of some of the phrases used will not be apparent until the phenomena in question have been considered in some detail.
[6]1µ is one-thousandth of a millimeter; 1µµ is one-thousandth of a µ, or one millionth of a millimeter.
[6]1µ is one-thousandth of a millimeter; 1µµ is one-thousandth of a µ, or one millionth of a millimeter.
It is recognized by all students of these matters that it is not possible to draw a sharp dividing line between these three types of conditions, and that they shade into each other, in many cases; but in general it may be said that a colloidal solution is one in which the dispersed particles are usually between 5µµ and 200µµ in diameter, are difficultly or not at all diffusible through the membrane of a simple dialyzer, cannot be filtered out of solution, do not settle out under the action of gravitation, and are visible only under the "ultramicroscope"; and one which has certain peculiar optical, osmotic, and other physical and chemical properties. Since colloidal particles are very minute in size, they possess very large relative surface areas as compared with their total mass or volume, very high surface tension, and a relatively high surface energy as compared with their total, or molecular, energy. These properties bring into play, in a substance which is in the colloidal condition, in a remarkable degree, all the phenomena which are associated with surface boundaries between solids and liquids, liquids and gases, etc.
The properties arising out of the colloidal condition are of such tremendous importance in connection with the vital phenomena exhibited by cell protoplasm that it is necessary to give some detailed consideration to them here. Many large volumes dealing with this condition of matter have been written, and it is very difficult to condense even the most important facts concerning it into a few pages, but an attempt has been made to present in this brief summary the most essential facts and principles involved in the colloidal phenomena.
Colloidal mixtures may exist in two different forms: one, in which the mixture is fluid and mobile, like a true solution, is known as a "sol"; and the other, which is a semi-solid, or jelly-like, form, is known as a "gel." Sols may be easily converted (or "set") into gels, by changes of temperature or of the electrolyte content, or by changes in the concentration of the mixture, etc., and in most cases gels can be converted again into sols. In some cases, however, gel-formation is irreversible, the gels are permanent and cannot be changed back again into sols by any known change in environmental conditions.
Depending upon whether the liquid dispersion medium is water, alcohol, ether, etc., sols are known as "hydrosols," "alcosols," "ethersols," etc.; and gels as "hydrogels," "alcogels," etc.
Sols in which the disperse phase is a solid are known as "suspensoids"; while those in which it is a liquid are "emulsoids." Thus, sols of most inorganic compounds, of dextrin, gelatin, and(probably) of casein, etc., are suspensoids; while sols of egg-albumin, of oils, etc., are emulsoids. The classification of these substances into suspensoids and emulsoids is, however, more a matter of convenience than of real difference in composition, since it is practically impossible to say whether many of the organic substances which normally exist in colloidal form are themselves liquids or solids, when in the non-dispersed form.
Suspensoids differ from mechanical suspension of solids in a liquid in that in the latter the solid particles settle toward the bottom of the mixture, because of the effect of the attraction of gravity upon them. The rate at which such particles settle depends upon the size and density of the particle and the viscosity of the liquid, and can be roughly calculated from the formula for Stokes' law for the rate of falling of a spherical body in a liquid. This formula is
2r2(s-s´)gV= —————9n
V= velocity of the falling body, in millimeters per second;r= radius of the particle, in millimeters;s= specific gravity of the solid;s´ = specific gravity of the liquid;g= the attraction of gravity, in dynes;n= the viscosity of the liquid.
For example, if this formula be applied to determine the rate at which the particles of gold of the size of those in a red gold sol would settle, if they were in mechanical suspension in water (r= 10µµ, or one-ten-thousandth of a millimeter;s= 19.3;s´ = 1;g= 980, andn= 0.01), it will be found that such particles will settle at the rate of approximately 0.0146 millimeter per hour, or a little over 10 mm. (0.4 inch) per month. Hence, the settling of such particles, if in mechanical suspension, would be measurable, although very slow. Shaking up thesuspensionwould cause the particles to rise through the liquid again. But in a gold sol, orsuspensoid, which contains particles of gold of the size used in this calculation, the gold particles do not settle, even at the slow rate as calculated above. They remain uniformly distributed throughout the liquid for an indefinite period or time. The reason for this phenomenon undoubtedly lies in the fact that these minute particles carry an electric charge, which, is of the same sign for all of the particles and results in a repellent action which keeps the particles in constant motion. This constant motion may easily be conceived to keep the particles uniformly distributed throughout the liquid, just as constant shaking would keep those of a mechanical suspension uniformly distributed through the mixture.
The sign of the electric charge on the particles of a sol may be either negative or positive, depending upon the chemical nature anddielectricconstants of the two phases of the system. The proportion of the total electric charge of the system which is of the opposite sign to that borne by the dispersed particles is, of course, borne by the liquid which constitutes the other phase. The origin of this electric charge on the colloidal particles is, as yet, not known with certainty; but it seems probable that it is due to a partial ionization of these small particles, similar to, but not so complete as, that which takes place when compounds which are soluble go into true solution in water, or other solvents which bring about the dissociation of dissolved substances.
The conditions necessary to bring a solid substance into a colloidal mixture with some liquid, or, in other words, to produce a suspensoid sol, require that the proportion of liquid to solid shall be large and some means of disintegrating the material which is to be dispersed into very fine particles. Many common chemical reactions, if carried out in very dilute solutions, result in the production of sols, especially if a small amount of some emulsoid is present in the reacting mixture; sols produced in this way are very stable, and the emulsoid which is used in stabilizing the sol is known as a "protective colloid." Direct methods of disintegration; such as reduction by chemical agents, discharge of a strong electrical current through the substance which is to be dispersed while it is submerged in the liquid, alternate treatment of finely ground material with alkali and acid so as to frequently change the electric charge, etc., are utilized for bringing inorganic compounds into the colloidal state.
Suspensoids usually contain less than 1 per cent of the solid dispersed through the liquid. In fact, extreme dilution is one of the necessary conditions for suspensoid-formation.
Emulsoids are much more easily produced than are suspensoids.The property of forming an emulsoid seems to be much more definitely a characteristic of the substance in question than does the formation of sols from solids which, under other conditions, may form true solutions. This difference may be due to the fact that the liquids which easily form emulsoids (usually those of organic origin) have very large molecules, so that the transfer from molecular to colloidal condition involves much less change in such cases than it does in the case of solid (inorganic) substances of relatively low molecular weight. This view of the matter is further borne out by the fact that solids which have very large molecules (generally of organic origin) take on the colloidal form much more readily than do those of small molecular size.
At the same time, a given liquid may form a true emulsoid when introduced into one other liquid and a true solution when introduced into another. Thus, soaps form emulsoids with water (true hydrosols); but dissolve in alcohol to true solutions, in which they affect the osmotic pressure, the boiling point of the liquid, etc., in exactly the same way that the dissolving of other crystalloids in water affects the properties of true aqueous solutions. Again, ordinary "tannin," when dissolved in water, produces a sol, which froths easily, is non-diffusible, etc.; but when dissolved in glacial acetic acid, it produces a true solution.
The concentration of the disperse phase may be much greater in the case of emulsoids than it can be in suspensoids. This is probably because the dispersed particles do not carry so large an electric charge and are not in such violent motion.
The one property which most sharply distinguishes sols from true solutions is their ability to "set" into a jelly-like, or gelatinous semi-solid, mass, known as a "gel," without any change in chemical composition, or proportions, of the two components of the system. In the gel, the two components are still present in the same proportions as in the original sol; but the mixture becomes semi-solid instead of fluid in character. Thus, an agar-agar sol containing 98 per cent of water sets into a stiff gel; while many other gels which contain 90 to 95 per cent of water can be cut into chunks with a knife and no water will ooze from them. The water is not in chemical union with the solid matter in the form ofdefinite chemical hydration, however, as the same gel is formed with all possible variations in the water content.
Gels may be either rigid, as in the case of those of silicic acid, etc., or elastic, as are those of gelatin, egg-albumin, agar-agar, etc. The latter are the common type of gels among organic colloids. They can be easily changed in shape, or form, without any change in total volume.
In gel-formation, the two phases of the system take a different relationship to each other. The disperse, or solid, phase becomes associated into a membrane-like, or film, structure, surrounding the liquid phase in a cell-like arrangement. That is, the whole mass takes on a structure similar to a honeycomb except that the cells are roughly dodecahedral in shape, instead of the hexagonal cylinders in which the bees arrange their comb cells, in which the original disperse phase constitutes the cell-walls and the original liquid, or continuous phase, represents the cell-contents. The cells of an elastic gel resemble closely the cells of a plant tissue in many of their physical properties. They are roughly twelve-sided in shape, as this is the form into which elastic spherical bodies are shaped when they are compressed into the least possible space.
Imbibition and Swelling of Gels.—When substances which are natural gels, such as gelatin, agar-agar, various gums, etc., are submerged in water, they imbibe considerable quantities of the liquid and the cells become distended so that the mass of the material swells up very considerably. This swelling will take place even against enormous pressures. For example, it has been found that the dry gel from sea-weeds will swell to 330 per cent of its dry volume, if immersed in water under ordinary atmospheric pressure; but that it will increase by 16 per cent of its own volume when moistened, if under a pressure of 42 atmospheres.
During the swelling of gels by imbibition of water, the total volume of the system (i.e., that of the original dry gel plus that of the water absorbed) becomes less. For example, a mixture of gelatin and water will, after the gelatin has swelled to its utmost limit, occupy 2 per cent less space than the total volume of the original gelatin and water. It has been computed that a pressure equivalent to that of 400 atmospheres would be necessary to compress the water to an extent representing this shrinkage in volume.
On the other hand, gels when exposed to the air lose water by evaporation, shrink in volume, and finally become hard inelastic solids, as in the case of the familiar forms of glue, gelatin, agar-agar, gum arabic, etc.
The difference in the relation of gels and that of non-colloidal solids to water may be illustrated by the different action of peas, beans, etc., and of a common brick, when immersed in water. Each of these substances, under these conditions, absorbs, or "imbibes," water; but the peas and beans swell to more than twice their original size and become soft and elastic, while the brick undergoes no change in size, elasticity, or ductility. In all cases of colloidal swelling, the swollen body possesses much less cohesion, and greater ductility, than it had before swelling. The essential difference in the two types of imbibition is that in the case of the non-swelling substances the cohesion, or internal attraction of the molecules of the material, is too great to permit them to be forced apart by the water; while in colloidal swelling, the particles are forced apart to such an extent as to make the tissue soft and elastic. It is possible, of course, to make this separation go still further, until there is an actual segregation of the molecules, when a true solution is produced; for example, gum arabic when first treated with water swells into a stiff gel, then into a soft gel, and finally completely dissolves into a true solution.
Reversibility of Gel-formation.—In some cases, the change of a sol to a gel is an easily reversible one. Glue, gelatin, various fruit jellies, etc., "melt" to a fluid sol at slightly increased temperatures and "set" again to a gel on cooling, and the change can be repeated an indefinite number of times. On the other hand, many gels cannot be reconverted into sols; that is, the "gelation" process is irreversible. For example, egg-albumin which has been coagulated by heat cannot be reconverted into a sol; casein of milk when once "clotted" by acid cannot again be converted into its former condition, etc. Irreversible gelation is usually spoken of as "coagulation." Some coagulated gels, by proper treatment with various electrolytes, etc., can be converted into sols, the process being known as "peptization"; but in such "peptized" hydrosols, the material usually exists in a different form than originally, having undergone some chemical change during the peptization, and the coagulation and peptization cannot be repeated, that is, the process is not a definitely reversible one.
Importance of Gel-formation.—From the physiological point of view, gel-formation is undoubtedly the most important aspect of colloidal phenomena. In the first place, the ability to absorb and hold as much as 80 to 90 per cent of water in a semi-solid structure is of immense physiological importance. In no other condition can so large a proportion of water, with its consequent effect upon chemical reactivity, be held in a structural, or semi-solid, mass. But a vastly more significant feature of the conditions supplied by the gel lies in the fact that the non-water phase, or phases, of the system are spread out in a thin film, or membrane, thus giving it enormous surface as compared with its total volume. This effect is easily apparent if one thinks of the enormous surface which is exposed when a tiny portion of colloidal soap is blown out into a "soap-bubble" several inches in diameter. This condition brings into play all the phenomena resulting from surface boundaries between solids and liquids, liquids and liquids, liquids and gases, etc., from surface tension, surface energy, etc. Among these effects may be cited those of adsorption, increased chemical reactivity due to enlarged areas of contact, permeability and diffusion, etc., the importance of which in the vital phenomena of cell-protoplasm will be discussed in detail in the following chapter.
Non-diffusibility.—The most characteristic property of all sols is the failure of the suspended particles to pass through a parchment, or any similar dialyzing membrane.
Visibility under the "Ultramicroscope."—The particles of a sol, in contrast with the molecules of a true solution, are visible as bright scintillating points under the ultramicroscope. This is a modification of the type of dark-field illumination of the ordinary microscope, as applied to microscopic studies, in which the solution to be studied is contained in a small tube or box of clear glass which is mounted on the stage of an ordinary microscope and instead of being illuminated from below by transmitted light is illuminated by focusing upon it the image of the sun, or of some other brilliant source of light such as an electric arc,by passing the rays from the source of light through a series of condensing lenses which are adjusted at the proper distance and angles to bring the image of the illuminating body within the tube containing the substance which is to be examined and in the line of vision of the microscope. Obviously, this results in intense illumination of any particles in the solution which come within this brilliant image of the sun, or arc, and therefore renders visible particles which are of less diameter than the wave-length of ordinary light (450µµ to 760µµ for the visible spectrum) and, hence, are not visible by the ordinary means of illumination in the direct line of vision. It will be apparent that what is seen in the field of the ultramicroscope is not the particles themselves, but rather the image of the sun (or other illuminating body) falling upon the particles which come within the image, just as one does not see the paper but only the image of the sun when the rays from the sun are brought to a focus upon a sheet of paper through any ordinary convex lens, or "burning glass." Hence, the ultramicroscope gives no idea of the shape, color, or size of the particles upon which the image falls; but it does permit the counting of the number of particles within a given area, and a study of their movements, from which it is possible, by mathematical computations, to calculate the relative size of the particles themselves. Repeated studies have shown that particles of the sizes between 5µµ and 250µµ in diameter, which are visible under the ultramicroscope, are sufficiently small to bring about the surface phenomena which are known as properties of colloidal solutions. Further, the ultramicroscope permits the observation of the growth, or disintegration, under various chemical reagents, of the individual colloidal particles, which appear as scintillating points in the field of the microscope; and the study of changes in relationships during gel-formation, peptization, etc.
The "Tyndall Phenomenon."—Colloidal solutions exhibit this phenomenon; that is, if a bright beam of light be passed through a sol which is contained in a clear glass vessel having parallel vertical sides, and the solution be viewed from the side, it appears turbid and often has a more or less bluish sheen. This effect is due to the small particles in the sol, of polarizing the light which is reflected from them, the blue rays being bent more than are those in the other part of the spectrum. The Tyndall phenomenon is similar in its effect in making the tiny particles of the sol visibleto the illumination of the dust particles in the air of a darkened room when a ray or narrow beam of light passes through it. In a true molecular solution, the particles are too small to be visible by this mode of illumination.
Other Optical Properties.—Sols are generally translucent and opalescent; many of them are highly colored, some of the sols of gold, platinum and other heavy metals possessing particularly brilliant colors. In general, metallic suspensoids are red, violet, or some other brilliant color; while inorganic suspensoids are bluish white, and emulsoids generally blue to bluish white.
Formation of Froth, or Foam.—Colloidal solutions, especially those of the natural proteins, fats, glucosides, gums, and the artificial soaps, have a strong tendency to produce froth, or foam, when shaken; this being due to the enormous surface tension resulting from the finely divided condition of the dispersed material.
Low Osmotic Pressure.—All colloidal solutions exhibit a very low osmotic pressure; the freezing point of the dispersion medium is lowered only very slightly and its boiling point is only very slightly raised by the presence of the dispersed particles in it.
Precipitation by Electrolytes.—Sols of all kinds are precipitated, or caused to form gels, by the addition of electrolytes, since these cause a disturbance of the electric charge on the dispersed particles, to which the colloidal condition is due. In the case of most emulsoids and of a few of the suspensoids, this change converts the mass into a stiff gel; but in that of many of the metallic suspensoids, the dispersed particles are gathered together into larger aggregates, which settle out of the liquid in the form of a gelatinous precipitate. In the latter case, the effect is usually spoken of as "precipitation" by electrolytes; while in the former, it is called "coagulation," or "gelation."
The effectiveness of the various electrolytes in bringing about this change is proportional to their valency; bivalent ions are from 70 to 80 times, and trivalent ions about 600 times as effective as monovalent ions.
Further, all sols in which the dispersed particles carry a charge of the opposite sign likewise precipitate both suspensoids and emulsoids.
A demonstration of the presence of an electric charge on the particles of a sol and a determination of its sign can be made byplacing the solution in a U tube, with a layer of distilled water above the sol in each arm of the tube, and then passing an electric current through the contents of the tube, keeping the electrodes in the distilled water, so that the migration of the particles toward one pole or the other can be observed by their appearance in the clear water at that end of the tube; or by passing an electric current through the observation chamber of an ultramicroscope, in which the solution under examination has been placed, and observing the migration of the particles across the field toward either one or the other (positive or negative) electrode.
Emulsoids and suspensoidsdiffer in their properties in the following respects. Suspensoids are always very dilute, containing less than 1 per cent of the dispersed solid; while emulsoids may be prepared with widely varying proportions of the two component liquids. Suspensoids have a viscosity which is only slightly greater than that of the liquid phase when it exists alone, and their viscosity varies with the proportion of dispersed solid which is present in the sol; while emulsoids have a very high viscosity in all cases. Emulsoids usually form stiff gels when treated with electrolytes; while suspensoids more commonly yield gelatinous precipitates under the same conditions.
Suspensoids and emulsoids which carry electric charges of opposite sign mutually precipitate each other. But emulsoids often protect suspensoids from precipitation by electrolytes, by forming a protective film around the particles of the suspensoids, which prevents the aggregation of the particles into the precipitate form.
If a sol be precipitated or coagulated by the action of an electrolyte, substances which may be present in solution in the liquid of the sol are carried out of solution and appear in the gel or precipitate. This phenomenon is known as "adsorption," which means the accumulation of one substance or body upon the surface of another body, as contrasted with "absorption," which means the accumulation of one substance within the interior of another. Since substances which are in the colloidal form have very large relative surface areas, it follows that the opportunity for surface adsorption on colloidal materials is very great.
Surface adsorption is a common phenomenon. It was extensively studied by the physicist, Willard Gibbs, who showed that adsorption will take place whenever the surface tension of the adsorbing body will be lowered by the concentration in its surface layer of the material which is available in the solution or other surrounding medium.
As applied to colloidal phenomena, adsorption may be exhibited in either one of four different ways, as follows: (1) A crystalloidal substance which is in solution may be adsorbed on the colloidal particles of a hydrosol, so that if the mixture be dialyzed, or filtered through a so-called "ultrafilter" (i.e., a filter with pores so small that it will retain colloidal particles) the dissolved crystalloid will remain with the separated colloidal particles, or the dissolved crystalloid will not react chemically as it would in a free solution. For example, if to a solution of methylene blue, which dyes wool readily, there be added a small quantity of albumin (a colloidal substance), the dye is adsorbed by the albumin and will no longer color wool with anything like the same readiness. (2) During gel-formation, electrolytes and other soluble substances which may be present in solution in the liquid may adsorbed out of the solution and appear in the gel. For example, a precipitate of aluminium hydroxide, or of silicic acid, is nearly always contaminated with the soluble salts which are present in the solution, and can be prepared in pure form only by repeated filtering, redissolving, and reprecipitating. (3) Colloidal substances may be removed from sols by being adsorbed upon porous materials like charcoal, fuller's earth, hydrated silicates, etc. For example, animal charcoal (or bone black) is used commercially for the clarification of sugar solutions, because it adsorbs out of these solutions the colloidal proteins, coloring matters, etc., with which they are contaminated. (4) Finally, colloids mutually adsorb each other, as in the case of the "protective colloids" previously referred to.
Certain characteristics of adsorption phenomena are of interest and importance from both the physiological and the industrial point of view. The following may be mentioned: (a)Amount of adsorption. Relatively more material is adsorbed out of dilute solutions than out of more concentrated ones. An increase of ten times in the concentration of the dissolved material results in only four times as much adsorption by the colloidal substance whichmay be introduced into the two solutions. In this, adsorption differs from chemical action, as the latter is proportional to the concentration of the reacting material which is present in the solution. (b)Adsorption out of different liquids, by the same adsorbing body, is different in amount. It is usually greatest out of water. Hence, many dyes may be adsorbed out of water by charcoal, porous clay, etc., and if the latter be then introduced into alcohol, or ether, the dye goes back into solution in these latter liquids. This process is often used industrially and in the laboratory for the purification of such substances when they are present in impure form in aqueous solutions. (c)Selective adsorption. Different substances are not adsorbed out of the same solvent to the same extent by the same adsorbing agent. Advantage is taken of this fact when filter paper is used in the so-called "capillary analysis" to separate different dyes, or other colloidal materials which have been stained different colors, into alternate layer by reason of the different rate at which the paper adsorbs the different materials out of the solution in which they are present together. (d)Similar relative adsorption by different adsorbing agents. Although different adsorbing agents may possess varying active surfaces and hence, variable adsorbing power, or rates of adsorption, they adsorb the same relative amounts of different materials; i.e., if substanceAadsorbs more ofXthan it does ofZout of any given solution, substanceBwill likewise adsorb more ofXthan ofZout of the same solution; although the actual amounts adsorbed byAmay be quite different from those adsorbed byB.