We can find many illustrations among chemical phenomena where one body, even though present in small quantity, acts as a go-between and makes possible an almost indefinite exchange of matter and energy. Take as an illustration, the part played by water in determining the explosion of oxygen and carbon-monoxide gas. Some years ago,Dixon46called attention to the fact that a mixture of these two gases when perfectly dry would not explode even by contact with red hot platinum wire. The presence, however, of a small amount of aqueous vapor would at once cause an explosion to occur. In confirmation of this observation,Traube47has reported that a flame of carbon-monoxide gas introduced into a perfectly dry atmosphere is at once extinguished. In a moist atmosphere, on the other hand, the flame will continue to burn indefinitely, that is as long as the CO gas is supplied. In these cases, the water, which is so necessary for the appearance of the reaction, and which furnishes a striking illustration of the action of a contact or catalytic substance is not purely passive. To be sure, only a minimal amount is necessary for the combustion of an indefinite amount of carbonic oxide, but the water enters into the reaction itself. It is to be noticed that carbonic oxide and water alone, even at high temperatures, will not react, but in the presence ofoxygen the water is decomposed with formation of hydrogen-peroxide, thus:
CO + 2 H2O + O2= CO(OH)2+ H2O2.
The hydrogen-peroxide thus formed combines with carbonic oxide to form carbonic acid, which in turn is decomposed into the anhydride CO2, with regeneration of water, the latter being available for further action of the same order:
H2O2+ CO = CO(OH)22 CO(OH)2= 2 CO2+ 2 H2O.
Indeed, as can be readily seen from the equations, this may be kept up indefinitely, a small amount of water,i.e., the go-between, the catalytic agent, sufficing to accomplish the transformation of almost any amount of carbon-monoxide. This, I think, furnishes an excellent illustration of the way in which catalytic agents, such as the proteolytic enzymes, may be supposed to act. It is truly contact action, but the agent is not purely passive; the enzyme combines with the substance undergoing proteolysis, and the resultant compound thus formed is enabled now to combine with water and undergo hydrolysis, something which could not be accomplished by the proteid and water alone, that is at body temperature. This new and more complex compound is naturally less stable and soon undergoes dissociation or cleavage with a splitting off of the original enzyme for one product, which is thus available for further action of the same order; while, as other products, we find the hydrated and otherwise altered substances coming from the proteid, and whose formation is the ultimate object of the whole process.
The parallelism between this hypothetical action of the proteolytic enzymes and the known reactions in the above combustion of carbonic oxide is certainly very close, andleaves little doubt that this explanation of enzyme action is, in a general way at least, correct. Thus the carbonic oxide, CO, brought in contact with pure, dry oxygen gas (apparently all that is necessary for its direct oxidation into carbonic acid, CO2), undergoes no change; the burning CO gas is at once extinguished. Evidently, something more is necessary in order to start the process of oxidation. So, too, in proteolysis; the process, as we shall see later on, is essentially one of hydration, but bring the proteid and the water, or acid-water, together and although all the conditions are apparently favorable for hydration there is, as you know, little or no change. But introduce the catalytic agent and immediately the reaction commences. In the case of the burning CO gas in contact with oxygen, the water acting as contact agent makes oxidation possible, enabling the main actors in the transformation to react upon each other. But, as we have seen, the contact agent is something more than a mere looker-on, it becomes for the time being an integral part of the molecule, undergoing change, combining with it and thus making possible the subsequent alterations characteristic of the specific transformation, in which, however, the regeneration of the contact agent is a prominent feature. So, too, with the proteolytic enzymes, pepsin and trypsin, they are the go-betweens, making possible the union of the proteids with water by combining with the proteid molecule and thus paving the way for both hydration and cleavage. In the cleavage of the complex molecule, we have the regeneration of the ferment as a prominent feature, and in proteolysis we understand that the regenerated ferment may act not only upon more of the original proteid, but likewise upon the primary products of its action, thus giving rise eventually to a row of more or less closely related cleavage products. Finally, we can conceive that the enzyme maygradually be affected by the process, that its regeneration may become less complete, and thus digestive power be eventually diminished.
Much more might be said in support of the above hypothesis. On the other hand, some objections might be raised against it, but I know of no more reasonable explanation of enzyme action than that here presented, or one which so well accords with all of the known facts concerning the conditions which modify proteolytic action.48Thus, the influence of heat, of the products of proteolysis, of acids, alkalies, and various organic and inorganic salts on the action of these digestive enzymes is such as lends favor to the above view rather than opposes it.
Proteidsare confessedly among the most complex bodies the physiologist has to deal with, while at the same time they are perhaps the most important, not only in view of their wide-spread distribution through animal and vegetable tissues, but because of the prominent part they take in the nutrition of the body. The more our knowledge is broadened concerning these varied substances, the more we are impressed with their complexity, and at the same time with the necessity for a more accurate study of both their composition and constitution. Concerning the latter, full fruition of our hopes is probably in the distant future, but every step of advance in this direction adds greatly to our resources in the interpretation of the varied and complex changes characteristic of proteid metabolism.Every study of proteid decomposition adds something to our store of knowledge, and gives perhaps an added fact available for broadening our deductions.49Moreover, the composition and general reactions of the proteids may be investigated with full confidence of obtaining many useful results, which must necessarily be an aid in interpreting the changes accompanying digestive proteolysis.
Take, for example, the single question of peptonization by gastric digestion. What is the nature of the process? Is the proteid transformed into a soluble and diffusible peptone as a result of hydration and cleavage, or is it a transformation which results from a simple depolymerization of the proteid molecule,i.e., are we to consider albumin and peptone as isomeric bodies? These questions, on which physiologists seem loath to agree, can certainly be answered definitely; not, however, by arguments but by careful experimentation, in which the composition of the proteid undergoing digestion must be a necessary preliminary factor, and the composition of the resultant product, or products, a secondary factor of equal importance. Further, the question needs to be answered not with reference to one proteid merely, but with reference to every proteid capable of digestion by either gastric or pancreatic juice. When these questions have been fully answered in this manner, we shall have positive data to deal with, and our conclusions will rest upon a foundation of fact not easily set aside. This is one of the problems upon which I have been at work for some years, and although progress may in one sense be slow, yet it is sure and gives results of no uncertain character.
First, then, let us consider briefly the nature of the proteids whose proteolysis we may be interested in; remembering, however, that in so doing we can merely touch upon the points essential for our purpose. Allow me to say in parenthesis that there is being published in Moscow a work on proteids alone of five volumes, 900 pages each, which it is supposed will constitute an exhaustive treatise of the subject.50
If we attempt to classify all of the proteid bodies hitherto discovered and studied we are at once confronted with a problem of no small proportions. So varied are they in their reactions, solubilities, and behavior toward general reagents, so inclined to merge into each other by almost insensible gradations that it becomes an extremely difficult matter to make an arrangement that will satisfy all the requirements of the case. I have to suggest, however, the following classification, which is merely a modification of several existing ones, based primarily upon chemical composition, and solubility in the more common menstruums.
Proteids may first be divided into three main groups as follows:
I.Simple Proteids.—Composed of carbon, hydrogen, nitrogen, sulphur, and oxygen, and yielding by decomposition aromatic bodies such as tyrosin, phenol, indol, etc.
II.Compound Proteids.—Composed of a simple proteid united to some non-proteid body.
III.Albuminoids.—A class of nitrogenous bodies related to and derived from proteids, but differing especially from the latter by great resistance to the ordinary solvents of true proteids.
The individual members of these three groups may be arranged as follows on the basis of solubility, coagulability, etc.:
I.Simple Proteids.—A.Soluble in water.—a.Coagulable by heat, and by long contact with alcohol. Albumins: serum-albumin, egg-albumin, lacto-albumin, myo-albumin, vegetable albumins.b.Non-coagulable by heat and by long contact with alcohol. Proteoses:51protoproteoses, deuteroproteoses. Peptones:51amphopeptones, antipeptones, hemipeptones.
B.Insoluble in water, but soluble in salt solutions.—a.More or less coagulable by heat. Globulins. 1. Soluble in dilute and saturated NaCl solutions. Vitellins. 2. Soluble in dilute NaCl solutions, but precipitated by saturation with NaCl. Myosins,paraglobulin52or serum-globulin, fibrinogen, myo-globulin, paramyosinogen, cell-globulins.b.Non-coagulable by heat, soluble in dilute NaCl solution and precipitated by saturation with NaCl. Heteroproteoses.
C.Insoluble in water and salt solutions, soluble in dilute alcohol—Zein, gliadins.
D.Insoluble in water, salt solutions and alcohol; soluble in dilute acids or alkalies.—a.Coagulable by heat when suspended in a neutral fluid. Acid-albumins, alkali-albumins or albuminates.b.Non-coagulable by heat when suspended in a neutral fluid. Antialbumids, dysproteoses, glutenins.
E.Insoluble in water, salt solutions, alcohol, dilute acids and alkalies; soluble in strong acids, alkalies, and in pepsin-hydrochloric acid and alkaline solutions of trypsin.—Coagulated proteids, fibrin.53
II.Compound Proteids.—A.Compounds of a proteid (globulin) with an iron-containing pigment, soluble in water and coagulable by heat and alcohol.Hæmoglobin, oxyhæmoglobin, methæmoglobin, etc.
B.Compounds of proteids with members of the carbohydrate group. Insoluble in water; soluble in very weak alkalies.—a.True mucins.b.Mucoids or mucinoids.
C.Compounds of proteids with nucleic acid. Phosphorized bodies yielding by decomposition metaphosphoric acid. Insoluble in water and in pepsin-hydrochloric acid, but more or less soluble in alkalies.—Nucleins.
D.Compounds of proteids with nucleins. Very soluble in dilute alkalies.—Nucleoalbumins, as casein of milk, and nucleoalbumins of cell-protoplasm and cell-nuclei, etc.
III.Albuminoids.—A.Soluble in boiling water with formation of gelatin and yielding by decomposition leucin and glycocoll.—Collagen (gelatin).
B.Insoluble in boiling water, and yielding by decomposition much leucin and some tyrosin, together with glycocoll and lysatin. Slowly hydrated by boiling dilute acids and by treatment with pepsin-hydrochloric acid.—Elastin.
C.Insoluble in water, dilute acids and alkalies, also in gastric and pancreatic juice. Yield leucin and tyrosin by decomposition.—Keratin, neurokeratin.
We may now advantageously consider the composition of a few of the more prominent representatives of the individual groups, taking for illustration those bodies which have been most thoroughly studied, and which we may have occasion to refer to in our discussion of proteolysis. I have not included in the table any of the alteration-products of the proteids formed by the action of pepsin-acid, trypsin, or boiling dilute acids, confining myself here simply to those bodies which occur ready-formed in nature.
Composition of Some of the More Prominent Proteids Occurring in Nature.*
Substance.CHNSOPAsh.Origin.Author.Serum-albumin63.056.8516.041.7722.29....0.57—Serum from horse bloodHammarsten.54Serum-albumin52.256.6515.882.2722.95....1.84Pleural exudationHammarsten.54Egg-albumin52.256.9015.251.9323.67........Non-coagulatedHammarsten.54Egg-albumin52.336.9815.891.8322.97....1.11Non-coagulatedChittenden and Bolton.55Lacto-albumin52.197.1815.771.7323.13........Cow’s milkSebelien.56Vegetable-albumin52.256.7616.071.4823.44....0.70Corn or maizeChittenden and Osborne.57Vegetable-albumin53.026.8416.801.2822.06....0.82WheatOsborne and Voorhees.58Proteose, animal52.136.8316.551.0923.40....0.79Hemialbumose, urineKühne and Chittenden.59Proteose, vegetable60.606.6816.331.6224.77....2.99Corn or maizeChittenden and Osborne.57Proteose, vegetable51.866.8217.32............0.25WheatOsborne and Voorhees.58Proteose, vegetable49.986.9518.78............1.80Flax-seedOsborne.60Proteose, vegetable46.526.4018.25............2.20Cocoanut meatChittenden and Setchell.61Vitellin, spheroidal51.716.8418.120.8522.48....1.20Corn or maizeChittenden and Osborne.57Vitellin, crystalline51.606.9718.801.0121.62....0.30Squash-seedChittenden and Hartwell.62Vitellin, amorphous51.816.9418.711.0121.53....0Squash-seedChittenden and Hartwell.61Vitellin, crystalline51.486.9418.600.8122.17....0.54Flax-seedOsborne.60Vitellin, spheroids51.036.8518.390.6923.04....0.49WheatOsborne and Voorhees.58Vitellin, crystalline51.636.9018.780.9021.79....0.56Hemp-seedChittenden and Mendel.61Vitellin, crystalline51.316.9718.750.7622.21....0.03Castor beanOsborne.63Vitellin, crystalline52.186.9218.301.0621.54....0.20Brazil nutOsborne.63Vitellin, semi-crystalline51.236.9018.401.0622.41....0.25Cocoanut meatChittenden and Setchell.61Myosin, 13 different samples52.827.1116.771.2721.90....1.45Muscle-tissueChittenden and Cummins.64Myosin, vegetable52.687.0216.781.3022.22....0.63Corn or maizeChittenden and Osborne.57Myosin, vegetable, crystalline52.187.0517.900.5322.34....0.10OatsOsborne.65Paraglobulin52.717.0115.851.1123.24....0.30Blood of horseHammarsten.66Fibrinogen52.936.9016.661.2522.26....1.75Blood of horseHammarsten.67Zein55.237.2616.130.6020.78....0.43Corn or maizeChittenden and Osborne.57Gliadin52.726.8617.661.1421.62....0.51WheatOsborne and Voorhees.58Gliadin53.016.9116.432.2621.39........OatsOsborne.65Glutenin52.346.8317.491.0822.25........WheatOsborne and Voorhees.58Coagulated proteid52.336.9815.841.8123.04....0.27Egg-albuminChittenden and Bolton.55Coagulated proteid51.586.8818.801.0921.65....0.25Vitellin, hemp-seedChittenden and Mendel.61Fibrin52.686.8316.911.1022.48....0.56Blood of horseHammarsten.67Oxyhæmoglobin53.857.3216.170.3921.84....0.43 Fe.Blood of dogHoppe-Seyler.68Oxyhæmoglobin54.717.3817.430.4819.60....0.39 Fe.Blood of pigHütner.69Mucin50.306.8413.621.7127.53....0.33From snailHammarsten.70Mucin48.846.8012.320.8431.20....0.35Submaxilliary glandHammarsten.71Chondromucoid47.306.4212.582.4231.28........CartilageMörner.72Nuclein50.607.6013.18........1.89....Human brainV. Jaksch.73Nuclein49.587.1015.02........2.28....PusHoppe-Seyler.74Casein52.967.0515.650.7122.780.84....Cow’s milkHammarsten.75Casein53.307.0715.910.8222.030.870.98Cow’s milkChittenden and Painter.76Nucleo-histon or leuconuclein48.417.2116.850.7024.412.42....LeucocytesLilienfeld.77Gelatin49.386.8117.970.7125.13....1.26Connective tissueChittenden and Solley.78Elastin54.247.2716.700.3021.79....0.90Neck-bandChittenden and Hart.79Elastin53.957.0316.670.3821.97....0.72AortaSchwarz.80Keratin49.456.5216.814.0223.20....1.01White rabbit’s hairKühne and Chittenden.81Neurokeratin56.997.5313.151.8720.46....1.35Human brainKühne and Chittenden.82Reticulin52.886.9715.631.8822.300.342.27Reticular tissueSiegfried.83
* Many of these results represent the average of a large number of individual analyses.
In considering the results tabulated above, it is to be remembered that all of these bodies, with the exception of keratin, neurokeratin, and reticulin, are more or less digestible in either gastric or pancreatic juice, or indeed in both fluids. I will not take time here to point out the obvious genetic relationships and differences in composition shown by the above data, but will immediately call your attention to the fact that there are other and more important points of difference between many of these proteids which are hidden beneath the surface, and which a simple determination of composition will not bring to light. I refer to the chemical constitution of the bodies, to the way in which the individual atoms are arranged in the molecule, on which hinges more or less the general properties of the bodies and which in part determines their behavior toward the digestive enzymes, as well as toward other hydrolytic agents. These differences in inner structure can only be ascertained by a study of the decomposition products of the proteids, and of the way in which the complex molecules break down into simpler. The nature of the fragments resulting from the decomposition of a complex proteid molecule, gives at once something of an insight into the character of the molecule. Thus, egg-albumin exposed to the action of boiling dilute sulphuric acid yields, among other fragments, large quantities of leucin and tyrosin, the latter belonging to the aromatic group and containing the phenyl radical. Collagen, or gelatin, on the other hand, by similar treatment fails to yield any tyrosin or related aromatic body, but gives instead glycocoll or amido-acetic acid, in addition to leucin, lysin, and other products common to albumin. Its constitution, therefore, is evidently quite different from that of albumin, but the composition of the body reveals no sign of it. Further, we have physiological evidence of this differencein constitution in that gelatin, though containing even more nitrogen than albumin, is not able to take the place of the latter in supplying the physiological needs of the body; its food-value is of quite a different order from that of albumin.
But while all of the individual proteids show many points of difference, either in composition, constitution, reactions, or otherwise, they are nearly all alike in their tendency to undergo hydrolytic decomposition under proper conditions; the extent of the hydrolysis and accompanying cleavage being dependent simply upon the vigor or duration of the hydrolytic process.
Furthermore, all of the simple proteids, at least, give evidence of the presence of two distinct groups or radicals, which give rise by decomposition or cleavage to two distinct classes of products. These two groups, which we may assume to be characteristic of every typical proteid, Kühne has named the anti- and hemi-group respectively. This conception of the proteid molecule is one of the foundation-stones on which rest some of our present theories regarding the hydrolytic decomposition of proteids, especially by the proteolytic enzymes. Moreover, it is not a mere conception, for it has been tested so many times by experiment that it has seemingly become a fact. The two groups, or their representatives, can be separated, in part, at least, by the action of dilute sulphuric acid (three per cent.) at 100° C. Thus, after a few hours’ treatment of coagulated egg-albumin, about fifty per cent. of the proteid passes into solution, while there remains a homogeneous mass, something like silica in appearance, insoluble in dilute acid, but readily soluble in dilute solutions of sodium carbonate. This latter is the representative of the anti-group, originally named bySchützenberger84hemiprotein,but now called antialbumid.85It is only slightly digestible in gastric juice, but is readily attacked by alkaline solutions of trypsin, being converted thereby into a soluble peptone known as antipeptone. In the sulphuric acid solution, on the other hand, are found the representatives of the hemi-group; viz., albumoses, originally known as one body, hemialbumose,86together with more or less hemipeptone, leucin, tyrosin, etc.
The fact that we have so many representatives of the hemi-group in this decomposition is significant of the readiness with which the so-called hemi-group undergoes change. All of its members are prone to suffer hydration and cleavage, passing through successive stages until leucin, tyrosin, and other simple bodies are reached. These, and other similar crystalline bodies, are likewise the typical end-products of proteolysis by trypsin, and presumably come directly from the breaking-down of hemipeptone. Antipeptone, on the other hand, is incapable of further change by the proteolytic ferment trypsin. Hence, the hemi-group can be identified by the behavior of the body containing it toward trypsin;i.e., it will ultimately yield leucin, tyrosin, and other bodies of simple constitution to be spoken of later on. The anti-group, however, will show its presence by a certain degree of resistance to the action of trypsin, antipeptone being the final product of its transformation by this agent;i.e., leucin, tyrosin, etc., will not result. In this hydrolytic cleavage of proteids the anti-group does not always appear as antialbumid. It may make its appearance in the form of some related body, the exact character of the product being dependentin great part upon the nature of the hydrolytic agent, but in every case the characteristics of the anti-group will come to the surface when the body is subjected to the action of trypsin.
The above-described treatment of a coagulated proteid with water containing sulphuric acid evidently induces profound changes in the proteid molecule. The conditions are certainly such as favor hydration, and in the case of complex molecules, like the proteids, cleavage might naturally be expected to follow. Analysis of antialbumid from various sources plainly shows that its formation is accompanied by marked chemical changes. Thus, the following data, showing the composition of antialbumid formed from egg-albumin and serum-albumin by the action of dilute sulphuric acid at 100° C., gives tangible expression to the extent of this change:
Egg-albumin.Antialbumid87fromegg-albumin.Serum-albumin.Antialbumid87fromserum-albumin.C .......52.3353.7953.0554.51H.......6.987.086.857.27N .......15.8414.5516.0414.31
In both cases there is a noticeable decrease in nitrogen, and a corresponding increase in the content of carbon. Evidently, then, this cleavage of the albumin-molecule into the anti-group on the one hand, and into bodies of the hemi-group on the other, is accompanied by chemical changes of such magnitude that their imprint is plainly visible upon the resultant products; changes which certainly are far removed from those common to polymerization.
This proneness of proteid matter to undergo hydration and subsequent cleavage is further testified to by the readiness with which even such a resistant body as coagulated egg-albumin breaks down under the simple influence of superheated water at 130° to 150° C. Many observations are recorded bearing on this tendency of proteid matter, but few observers have carried their experiments to a satisfactory conclusion. A recent study of this question in my own laboratory, has given some very interesting results.88Thus, coagulated egg-albumin placed in sealed tubes with a little distilled water and exposed to a temperature of 150° C. for three to four hours, rapidly dissolves, leaving, however, an appreciable residue. The solution reacts alkaline, there is a separation of sulphur, and in the fluid is to be found not albumin, but two distinct albumose-like bodies, together with some true peptone, and a small amount of leucin, tyrosin, and presumably other bodies.89The albumose-like bodies are in many ways quite peculiar. In some respects they resemble the albumoses formed in ordinary digestion; but in others they show peculiarities which render them quite unique, so that they merit the specific name of atmidalbumoses, as suggested by Neumeister. What, however, I wish to call attention to here is the composition of these albumoses. Prepared from coagulated egg-albumin by the simple action of heat and water, they show a deviation from the composition of the mother-proteid, which plainly implies changes of no slight degree. This is clearly apparent from the following table: