ACTION OF ENZYMES.

In a recent work,94 Dr. Bayliss defines enzymes as the “catalysts produced by living organisms.” A catalyst is a body which greatly accelerates the rate of reaction in chemical processes, without apparently taking part in the process. For instance, peroxide of hydrogen is decomposed into oxygen and water by mere contact with finely divided platinum, while the latter remains unaltered in the process. In this case the platinum black is the catalyst.95

In natural processes the best known type of an enzyme is diastase (amylase), the enzyme contained in malt, and which enables the malt to convert starch into dextrin and sugar (maltose). It is capable of transforming more than 2000 times its own weight into sugar, which fact is quite sufficient to show that its action differs from that of an ordinary chemical reaction. Another enzyme, sucrase, according to O’Sullivan and Thompson, will hydrolyze 100,000 times its weight of cane sugar to invert sugar. Rennet will coagulate 250,000 times its own weight of casein in milk. The list of enzymes grows longer almost daily, as some new one is separated having a specific action, until one is almost led to believe that the mechanism of life itself, as manifested in the cell, is due to enzymes.

It has been found that enzymes act very much in the same way as inorganic catalysers. As an example, the velocity of the reaction of invertase (the enzyme of yeast which hydrolyses cane sugar to grape sugar) has been compared with the same hydrolysis brought about by heating a solution of cane sugar with a mineral acid. In both cases the reaction is in accordance with the law of mass action (Guldberg and Waage) that the amount of sugar transformed will decrease as less remains to be transformed. In the diagram (Fig.26) the curve A is for invertase (Jas. O’Sullivan, “Journ. Inst. of Brewing,” vol.v. p.168); curve B is for the hydrolysis by acid (Wilhelmy), from which it will be seen that the manner in which the hydrolysis proceeds is practically the same in both cases.96

In the case of fermentation by the living organism, the fermentation rises rapidly, and then gradually slows down and comes to an end before the whole of the fermentable matter is used up; the curve, therefore, is of a hyperbolic character, C in the diagram, which represents, in a general way, the fermentation of glucose by B. furfuris. The ordinates represent the amount of acid produced by the bacteria. The time in this case would be 176 more nearly represented by hours instead of minutes on the abscissa.

The mathematical expression for the velocity of the reaction is

dx/dt = k(a - x)

where

a = original concentration of solution
x = the quantity transformed in time t
k = coefficient of velocity of the reaction

By integrating the above equation, it may be shown that

1/t log a/a – x = k

For the experiment of J. O’Sullivan with invertase, k has a mean value of 0·0013; for Wilhelmy’s experiment with acid, k has a mean value of 0·001377.

Enzymes are produced by the living cell, with other secretions, and as a consequence are found in all plants and animals. Certain organs, however, produce enzymes in large quantities, or appear to be specially set apart for their production. In plants, the seeds are the chief seat of enzyme activity; in animals, certain glands, such as the salivary glands and the pancreas. The mucous membrane of the stomach and intestines also secrete enormous quantities.

The production of enzymes by bacteria was observed by Wortmann in 1882. It has been found that the secretion of the enzymes depends upon the composition of the nutrient medium in which the bacteria are grown. For instance, Pfeffer found that the secretion of diastase by Bacillus megatherium depended upon the amount of cane sugar in the nutrient medium. The cane sugar checked the secretion of the diastase, and the same effect was observed in the case of the common mould Penicillium glaucum. In Bacillus mesentericus vulgatus, diastase has been found to exist side by side with four other enzymes. Passini,97 in studying the putrefactive anaerobic bacteria of the normal human intestines, succeeded in separating from B. putrificus a proteolytic enzyme filtered free from bacteria, and which caused proteolysis in media which were too acid to permit of the bacteria growing. The enzyme easily dissolved, without previous neutralization, the coagulated casein caused in milk by old coli cultures. Acid gelatin media were also liquefied by the enzyme.

From the evidence we have at present it seems probable that every variety of enzyme, hydrolysing, oxidizing, ammoniacal, etc., can be produced by bacteria.

The following useful classification of enzymes is due to Effront.98

A more recent classification based on chemical properties is that of Kossel and Dakin.99 They divide ferments into two classes:—

Group 2 is sub-divided into—

Although it is somewhat doubtful whether the enzymes contained in dog dung are of glandular origin,100 it is quite certain that other enzymes are secreted by bacteria developing in the dung while it is kept prior to being used for puering. These enzymes may be separated by the following method. About 150c.c. of puer is well mixed with an equal quantity of glycerin, and allowed to stand for seven days. It is then filtered through paper by means of a pump, and yields a clear filtrate of a deep golden-brown colour; the filtrate is poured in a thin stream into a tall vessel containing about 1500c.c. of 98 per cent. alcohol. A copious flocculent precipitate of the albuminous matter and enzymes is thrown down, the solution is filtered and the precipitate washed on the filter with absolute alcohol, and then dried over sulphuric acid in vacuo. The resulting powder is, of course, a mixture of all the albumins in solution, and probably only a small portion of it consists of the pure enzymes. We have merely succeeded by this method in concentrating them. The property of albuminous bodies in the act of coagulation to carry down soluble matter is well known, and this also renders the preparation of any pure proteid extremely difficult. It may be mentioned here that recent evidence goes to show that enzymes are not of a proteid nature, since, by repeated purification, the proteid matter may be almost entirely got rid of, while the activity of the residue containing the enzyme becomes considerably greater.

Krawkov’s method of preparing diastase from saliva (Green, p.46) may also be mentioned, as it is of general application. It consists in salting out the enzymes, by saturating the clear solution (in this case saliva diluted with an equal volume of water) with neutral ammonium sulphate. The precipitate which is caused by the saturation is collected on a filter, and washed for a short time with strong alcohol. It is then allowed to stand under absolute alcohol for one or two days, and finally dried at 30°C. On extraction with water it yields a solution which is strongly diastatic, and which gives no proteid reactions.

There are several other methods for the preparation and purification of enzymes, but up to the present it may safely be stated that no one has succeeded in preparing an enzyme in a state of purity.

In considering the mechanism by which enzymes act, it must be remembered in the first place that they are colloids, and, as such, will form absorption compounds with the substrate, or body, upon which they are acting. It is difficult to understand how an enzyme can exert an action on the substrate, unless it enters into some kind of combination with it, although this may be only a temporary one. The action of some enzymes has been found to be due to extremely small amounts of certain metals, e.g. in the case of the oxidizing enzyme laccase, the metal is manganese. In the purest samples of this enzyme prepared, 0·16 per cent. Mn was found, and it has been supposed by some observers that the whole of the action of the enzyme may be attributed to the physical state of the manganese which it contains. According to this hypothesis,101 the active part of the enzyme is the ion Mn. This ion may exist in the solution in two conditions, differing by the electric charge which they carry. One of them Mn₊₊ carries two positive charges, the other Mn₊₊₊ carries three. In the first phase, Mn₊₊ is transformed into Mn₊₊₊ by absorbing the charge of one ion of hydrogen (H₊), and two hydrogen ions thus discharged, in the nascent state, unite with the oxygen dissolved in the liquid to form water.

2Mn₊₊ + 2H₊ + 0 = 2Mn₊₊₊ + H₂O

In the second phase the ion Mn₊₊₊ with three charges will be transformed into the ion with two charges, by decomposing a molecule of water, of which the nascent oxygen will attach itself to the oxidizable body R yielding the oxide RO.

2Mn₊₊₊ + H₂O + R = 2Mn₊₊ + 2H₊ + RO

and the same cycle of operations will begin again, and continue indefinitely.

In the above illustration the enzyme action was an oxidizing one. In the case of puering it is a hydrolytic action, in other words a molecule of water is added to the skin substance. The fibre or some portion of it is converted first into proteoses, and finally into peptones, and simpler bodies. The active metal in these cases appears to be calcium, but by what mechanism it brings about the hydrolysis is at present unknown.

Pozerski102 found that the pancreatic juice, which is secreted after injections of certain sera (anti-pancreatic action), and as a consequence has no pancreatic action, contains no calcium, but pancreatic juice secreted under the influence of pilo-carpin is more or less rich in calcium, and its proteolytic action increases about equally with the amount of calcium contained in it. The same probably holds good for the intestinal juice.

Victor Henri has shown that the power of metals in the colloidal state to bring about these catalytic actions varies with the metal employed, and is in inverse ratio to the size of the particle. There is a very interesting and wide field of research open here in order to determine the conditions under which the various metals act. To this end the ashes from the purest enzyme preparations might be studied, and methods devised for producing these metals in the colloidal state, for it seems evident that it is the state of the body acting which gives it the properties observed, and not its chemical properties in the usual sense.

The enzymes contained in dog dung which are effective in the puering process belong to several groups, principally the proteolytic and lipolytic groups, but indirectly enzymes of the first group (see p.132), (fermenting carbohydrates) and of the fifth group (fermenting urea) also play some part by decomposing various compounds (e.g. cellulose and urea) contained in the puer.

The action of certain enzymes from the animal body upon skin has been tried by the author.103 Those selected were pepsin and pancreatin,104 as being most likely to be present in dung. Pepsin only acts in presence of HCl. Two portions of the same skin were taken, one of them was treated with a 1 per cent. solution of pepsin, acidified with 0·2 per cent. of hydrochloric acid; the other in a bate liquor of dogs’ dung (puer), both at a temperature of 40°C. At the end of one hour the skin in the pepsin solution was considerably “fallen,” but that in the manure solution was bated nearly away, i.e. the greater part of it was dissolved. A 1 per cent. solution of pancreatin (Mercks) was found to act far more rapidly than pepsin; 1·5 per cent. of chloroform was added to the solution, to prevent the development of bacteria. The skin was reduced, but had not the peculiar touch of a puered skin. As will be shown later, this was found to be due to the absence of any chemical action upon the lime salts in the skin, and consequently it felt “limey.” This action took place in the puered skin, but not in the skin treated with pancreatin alone.

W.J. Salomon (19) has also attributed the activity of the bate to pepsin and pancreatin, but he does not give any proof of the presence of these ferments in the bate.

Since it is practically impossible up to the present, to separate these enzymes from the dung in a state of purity, the method described on p.134 was employed.

The enzymes prepared in this way consist of a mixture of all the enzymes present in the dung, the amount obtained from 1000grm. of dung being about 4grm. The product had a slight diastatic action upon starch; 0·5grm. in 100c.c. of water at 35°C. was found to have a very considerable reducing action upon skin, and when combined with the amine compounds prepared from the dung, the action was more powerful, and more rapid than with puer. Limed skin was puered in thirty minutes in this solution to a perfect condition, in the absence of bacteria, and with no evil smelling compounds. The reaction of the solution at the beginning of the experiment was faintly alkaline; at the end of the experiment it was considerably alkaline.

This experiment proves that the action of the dung is a complex one, due to the combined action of enzymes and chemical compounds upon the skin. These compounds, which are principally amines, and salts of amino-acids, probably assist the enzymes, and at the same time act upon the lime remaining in the skin from the previous liming process. Whether a skin, which has never been submitted to the liming operation, could be bated by enzymes alone, without the addition of amines, has not, so far as I know, been tried, but it is highly probable that this would be the case.

In order to compare the action of the enzymes prepared from dung with that of the enzymes produced by bacteria, a mixed culture of bacteria from puer in dextrose gelatin, after seven days’ growth, was taken. 200c.c. of this was mixed with 200c.c. of dilute alcohol (65 per cent.), and well shaken: gelatin and albuminoid bodies are by this means precipitated. The liquid was filtered and poured into eight times its volume of 98 per cent. alcohol. The precipitate which came down was washed with absolute alcohol, and dried in vacuo. The enzymes thus obtained were re-dissolved in water, and the former experiment with the skin repeated with this solution, with the addition of the amines. The skin was brought down in exactly the same way as before, showing conclusively that it is the enzymes produced by bacteria, acting in conjunction with the amines, which bate the skin. It would seem that the action of the enzymes is aided by the presence of amine compounds, in addition to the chemical action which these latter have upon the skin. The action is interdependent, i.e. bacterial action alone is insufficient, and chemical action alone is insufficient, the true bating action being a combination of the two.

There seems little doubt that it is the enzymes which dissolve the skin substance, or rather certain parts of the intercellular substance of the fibres, and the compound of this substance with lime. The action is a digestive one, and may be compared, as we have shown, to that of the digestive ferment of the pancreas.

This fact has been made use of in the artificial bate “Oropon” (see Chapter VII.), in which extract of pancreas is combined with ammonium chloride, and some inert material.

The effect of puering on the fatty matter in the skin is well known. The fat and grease are partially emulsified, and set free, so that they can be removed by scudding. This action is a most important one, and one in which artificial bates have hitherto been wanting. The emulsification of the fats is brought about by means of an enzyme either identical with, or closely resembling, lipase.105 This enzyme is found in the pancreatic juice, and in the seeds of many plants. It brings about the emulsification of the fat by saponifying a portion of it, i.e. the fat is split up into glycerin106 and a fatty acid, according to the equation—

C₃H₅(C₁₈H₃₅O₂)₃ + 3H₂O
Stearin.
= C₃H₅(OH)₃ + 3(C₁₈H₃₅OHO)
Glycerin. Stearic Acid.

Lipase was one of the first enzymes in which the reversibility of the reaction was shown, i.e. it is not only capable of hydrolysing a fat, but also of causing the formation of one by the combination of the fatty acid and glycerin.107 This explains why the reaction of such an enzyme is never complete. An equilibrium is reached just as with ordinary reversible chemical reactions like the precipitation of magnesium hydroxide by ammonia.

Lecithin, and possibly other fatty compounds, are known108 to be important auxiliaries in the ferment-like actions produced by toxins; cholesterol has a similar effect, and as this body is a constituent of dung, it may play some part in puering. Here, again, is a further problem awaiting investigation.

Loevenhart has shown that the bile salts, sodium cholate, and sodium glycocholate, greatly increase the activity of lipase, and Magnus found that synthetic bile salts have the same effect; such bodies are known as co-enzymes. I have shown that bile itself is not favourable to the bating action, but the bile salts, by stimulating the action of the fat splitting enzymes, are probably essential to the full action of the dung bate.

Another enzyme which may be of importance in puering is erepsin, the enzyme of the intestinal juice, which is responsible for the completion of the digestive process. The pancreatic enzymes act upon the peptones produced by the pepsin of the stomach, splitting them up into simpler compounds, while the erepsin acts further upon these products. It dissociates albumoses and peptones into amino acids, taking as it were the last traces of nutriment from the food passing through the intestine. It acts best in alkaline solutions.

This ferment is very widely distributed in the animal kingdom, and occurs in other organs and tissues besides the intestines. The quantity of erepsin in the fresh fÆces must be considerable, since a dog secretes from 400 to 500c.c. of intestinal juice per day. It remains to be shown whether this retains its properties after excretion, and for how long.

A most important point in connexion with the activity of enzymes is the reaction of the medium, i.e. its acidity or alkalinity, or, more strictly speaking, its hydrogen ion concentration. A very slight increase or decrease of the acidity or alkalinity of the liquor will diminish the rate of action of the enzyme by a large amount, and in some cases cause the action to cease. In all cases enzymes have an optimum acidity or alkalinity; in other words, for every enzyme there is a particular hydrogen ion concentration at which its activity is at a maximum. The work of Soerensen, to which I have already referred, gives a very complete account of this aspect of enzyme action and also of the methods he employed to investigate it. His work should certainly be carefully studied by anyone wishing to take up this part of the subject.

The same remarks as to optimum conditions apply to temperature, although the effect of this is better known. Most tryptic enzymes act best at the body temperature, viz. 98° to 100°F. (37° to 40°C.), and hence puering should be conducted at this temperature. In the case of the hen or pigeon-dung bate the enzymes have not yet been studied, so far as I am aware, so that it is not possible to give an account of them. At the same time it seems highly probable that, as these bates are employed at comparatively low temperatures, the enzyme action is kept back, and therefore the chief action would be a chemical one.

Enzymes are retarded in their action in the first place by their own products, in a very similar manner to bacteria, in the second place the retarding or inhibiting action is brought about by the so-called anti-bodies. Of these the longest known and best studied are those which are produced by toxins, and which neutralize the action of the toxins upon the animal organism (anti-toxins).109 Normal serum also inhibits the action of trypsin and many other enzymes. Another very important group of anti-bodies are the precipitins. If the serum of an animal be injected repeatedly into another animal of different species, a precipitin appears in the serum of the animal treated, which causes a precipitate when added to the serum of the first animal. The special importance of this fact is, that it can be utilized as a method of distinguishing between human blood and that of animals, which is often of importance in medical juris-prudence.110

This fact has been made use of to distinguish dog dung from other matters, with which it has been adulterated.111 A perfectly clear extract of dog dung filtered free from bacteria was injected into a rabbit. The serum obtained from the rabbit was found to contain a precipitin, and on addition of the serum to the dog dung extract a precipitate was produced. When the serum was added to the extract from the dung of another animal no precipitate was formed. When added to the extract of the adulterated dung a very much smaller precipitate was produced than with the extract from dog dung alone.

The coli bacteria in the puer also produce an anti-body—agglutinin.112 If a culture of B. coli be examined under the microscope, the bacteria are seen moving here and there in the liquid, evenly distributed. On the addition of a trace of the serum of an animal which has been previously injected with coli bacteria the bacteria on the slide cease their movements and collect together in clumps. They are said to be agglutinated. This property is used to diagnose bacteria in suspected cases of cholera and some other diseases.

The wonderful discoveries that have been made in this direction constitute one of the most marvellous chapters in the history of science. Serum diagnosis and serum therapeutics are now firmly established as invaluable aids to the physician in his fight against disease and death.

The extent of our present knowledge of the action of enzymes in puering may be summed up as follows. Active enzymes are produced by bacteria growing in the infusion of dung, in addition to digestive enzymes which may be originally present in the dung; the bacterial enzymes are produced more rapidly in a dilute infusion, as employed in the puer wheel, than in the dung itself. The enzymes are of various kinds, proteolytic, peptolytic, lipolytic, etc., but the proteolytic and lipolytic are the most important. These have a solvent action on the fibres of the skin, but little or no action on the hyaline layer, at the concentration usually found in the puer liquor. The fatty matters and soaps in the skin are acted upon by the lipolytic enzymes, and the fats to some extent emulsified, so that they may be easily removed from the skin by scudding or pressing.

It must be clearly understood that enzyme action alone is not sufficient, as has been previously explained, but that the dung enzymes, acting in conjunction with the chemical compounds present, produce the specific puering effect.

There is still much work to be done before the action of the enzymes in dung is fully understood, but from the above short account it will be seen that the part they play in the bate is of great importance.

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