Laboratory, St Thomas's Hospital.)

Transcription

1 THE COAGULATION OF MILK BY RENNIN. BY JO H N MEL L A N B Y, M.D., Lecturer in Physiology St Thomas's Hospital Medical School. (From the Physiological Laboratory, Cambridge, and the Physiological Laboratory, St Thomas's Hospital.) CONTENTS. PAGE Introduction and methods.345 Coagulation of milk by gastric rennin and pancreatic rennin 347 (a) Rennin variable. (b) Caseinogen variable. (c) Calcium variable. (d) Volume variable. A comparison of the effects of calcium, barium, strontium, and magnesium chlorides.350 The adsorption of rennin by the coagulum, and by extraneous proteins.352 The presence of anti-gastric rennin and anti-pancreatic rennin in serum.353 The electrical state of milk during rennin coagulation Discussion of results..355 Summary 361 Introduction. The work of Hammarsten(i) on the coagulation of milk by rennin showed that in the formation of casein from caseinogen a double process is involved-the rennin converts the caseinogen into paracasein or soluble casein; the calcium salts present in the milk, precipitate the paracasein as casein. These statements have been confirmed by subsequent workers. But recent work on the coagulation of milk has been dominated by the hypothesis put forward by Pavlov(2) in a preliminary communication iu 1901, and published in detail by Pavlov and Parastschuks in 1904, that rennin and pepsin are identical. This hypothesis was based upon the ubiquitous nature of the rennin enzyme, its constant occurrence with a proteolytic enzyme, and the similarities of its resistances to destructive agents with those shown by pepsin.

2 .346 J. MELLANBY. In 1901, and independently of Pavlov, Nencki and Sieber(4) put forward the hypothesis that the milk clotting and proteolytic properties of gastric juice are due to different side chains of the same molecule. Pavlov's hypothesis, that the milk coagulating property of gastric juice is due to pepsin has been supported by the experimental work of Winogradow(ro, Gewin(6), Sawjaloff(7), Jacoby(s), van Dam(s), Funk and Niemann(io), and Michaelis(ii). But the hypothesis as to the identity of gastric rennin and pepsin has not been generally accepted. Hammarsteno2) regards the two ferments as distinct and separate and has brouight forward a mass of evidence in favour of his contention. Among other observations he finds that both pepsin and rennin are precipitated by magnesium carbonate or lead acetate but subsequently rennin only is soluble; that when a mixture of rennin and pepsin is heated to 40c C. rennin is more rapidly destroyed than pepsin and pepsin ultimiately remains; that an extract of calves' stomach readily coagulates milk whilst an extract of dogs' stomach does not, and that both peptic and coagulating effects are quite different. Hammarsten's views are supported by the experimental evidence of Rakoczy(ia), Taylor(l4), van Hasseltal5), and Porter(16). It has been recognised for some time that not only gastric juice but also pancreatic juice coagulates milk. Any hypothesis, therefore, which may be adopted from experiments on the coagulation of milk by gastric rennin should be capable of being extended to the coagulation of milk by pancreatic rennin. Kuhne pointed out that extracts made from the pancreas of a dog caused milk to coagulate. Sir William Robertsl7) investigated the paracasein reaction of milk. Edkins(18 determined the influence of various conditions on this coagulation. Brodie and Halliburton(o9) investigated the whole process in detail using pancreatic juice obtained from fistulae in dogs. As a result of their observations they concluded that the coagulation of milk by pancreatic juice is essentially different to that caused by gastric juice. Vernon(2o) investigated Roberts paracasein reaction of pancreatic juice and concluded that this reaction depends upon pancreatic rennin and not upon trypsin. Methods. In the following experiments two main problems have been investigated: the ultimate mechanism involved in the coagulation of milk by rennin, and the nature of rennin. The experiments detailed were done on fresh milk and comparative experiments were always done on the same specimen of milk. The

3 RENNIN ANJD MILK. 347 gastric rennin used was obtained by dissolving Armours scale pepsin in water, or by diluting a glycerine extract of pepsin. Both these preparations were extraordinarily efficient in coagulating milk under optimal conlditions. For pancreatic rennin a glycerine extract of the pancreas was used. 1 c.c. of a solution obtained by diluiting this glycerole of pancreatin with one hundred volumes of water was an efficient coagulant of 3 c.c. of suitably prepared milk. The times of coagulation of the milk were determined by noting the first appearance of discrete particles. When the coagulation times were comparatively short (under ten minutes), thie appearance of these discrete particles gave a particularly sharp endpoint; with longer coagulation times the endpoint was not so well marked, but in these cases the comparative error was not of such a magnitude as to obscure the significance of the experimental results. THE COAGULATION OF MILK BY GASTRIC RENNIN AND PANCREATIC RENNIN. The effects produced by varying conditions on the coagulation of milk by gastric rennin and pancreatic rennin are considered together in order that the fundamental similarity of the milk coagulating process caused by these two rennin enzymes may be appreciated. Pancreatic rennin has not been generally recognised and it may be useful to state the conditions under which this ferment manifests itself. V. J. Woolley(2l) and I showed that a glycerine extract of fresh pancreas or pancreatic juice itself has no coagulating action on milk. Dilution with water of a glycerine extract of the pancreas or addition of enterokinase to pancreatic juice develops this power in them. Again the milk coagulating property of activated pancreatic juice is not so evident as in gastric juice since milk contains too -little calcium salt for pancreatic rennin to produce a typical coagulum in it. But milk to which calcium chloride has been added coagulates in a typical manner after the addition of minute quantities of activated pancreatic extracts. In the coagulation of milk by rennin three substances are essential, caseinogen, rennin and a calcium salt. The effects of varying these factors on the coagulation process are shown in the following experiments. (a) Rennin variable. Winogradow(5) showed the inverse proportionality which exists between the coagulation time of milk and the

4 348 J. MELLANBY. amnount of gastric rennin added. This observation was confirmed by Fuld(2). The following figures show the relation between the coagulation time of milk and the amounts of gastric and pancreatic rennin added. The milk to which the gastric rennin was added bad been diluted with an equal volume of CaCl -O5N; the milk to which the pancreatic rennin was added, had been diluted with an equal volume of CaC12 IN. (The calcium was increased in amount to ensure coagulation.) Gastric rennin Coag. time Pancreatic rennin Coag. time 05 C.C. 30 mins. *15 c.c. 60 mins. *1,, 6,, *2,, 17 *2,, 4i,, *25,, 8,,.4,, 2.3,, 4 *'6,,t14,, *4,, 3 *8,, 50 sees. *6,, 14,, 1,0,, 40,, 8,, 1 Pancreatic rennin coagulates milk as readily as gastric rennin provided the calcium content of the milk be raised to an adequate -amouint. Further the same law holds for the two coagulations-with large amounts of rennin and quick coagulation times the time of coagulation is universely proportional to the amount of rennin added; with small amounts of rennin this proportionality is lost the times of coagulation being indefinitely extended. (b) Caseinogen variable. Precisely similar results are obtained when the amount of caseinogen is varied and the amount of rennin added is kept constant. These experimeuts were made by determining the quantity of calcium in the milk, and, on diluting the milk in order to vary the caseinogen, the requisite amount of calcium required to keep the percentage of calcium constant, was added. The volume was constant in every case. Coag. by I2 c.c. of gastric rennin Coag. by *2 c.c. of panc. rennin Milk Ooag. time Milk Coag. time 1 c.c. 3 mins. 1 c.c. 1 min. 2,, 5,, 2,, i 3,, 7i,, 3,, 2 it 4,, 11,, 4,,3 5,, 15i, 5i,, *, 6,, 33,, 6,, 14 7,, no coag. in 1 hr. 7,, 50 From the above figures it is clear that the coagulation time of milk can be varied not only by altering the amount of rennin but also by

5 RENNIN AND MILK. 349 altering the amount of caseinogen-in fact that the coagulation times in the two cases are fundamentally similar. The results indicate that a definite union takes place between the rennin and caseinogen, and that the time of coagulation of milk is dependent upon the quantities of rennin and caseinogen involved in this union. (c) Calcium variable. Reichel and Spiro(2s) extended Winogrado w's observation as to the reaction velocity of milk coagulation and showed that a similar inverse proportionality exists between the time taken by a definite quantity of rennin to coagulate milk and the amount of calcium present in the milk. The dependence of the coagulation time of milk upon the amount of calcium present is well shown in the following figures. Coagulation by 4 c.c. gastric rennin Coag. by 1 c.c. panc. rennin CaC12 In milk Coag. time CaCI2 in milk Coag. time *01 N 7i mins. *026 N 29 mins. 03 N 2,, 033N 5 05N 1,, 039N 07 N i046 N The effect of altering the calcium content of milk in the coagulation time is similar to the effect observed on altering the amount of rennin. (d) Volume variable. In the following experiments in every case the quantity of caseinogen and rennin was the same but the volume varied, the change in volume being so made that the percentage of calcium in the fluid was kept constant. Volume Milk CaCI2 (,2 N) Gastric rennin (*1 %) Coag. time 10 c.c. 5 C.c. 2 c.c. *2 C.C. 1' 20" 15,, 5,, 3,, I2,, 1,15" 20,, 5,, 4, *2,, 1' 20" 25,, 5,, 5,, 2,, 1' 25" The constancy of the time of coagulation under these conditions affords strong evidence that in the coagulation of milk the gastric rennin and caseinogen are chemically related to one another whilst the calcium salt acts by virtue of the charge on its ions. The same striking result was observed when milk was coagulated by pancreatic rennin under the same conditions. Volume Milk CaCl N Pancreatic (1:10) Coag. time 2 c.a. 1 a.c. -9 c.c. 1 c.c. 4 mins. 3,, 1, 1-9,,1,), 3*, 4,, 1,, 2'9,, 1,I 3, 5,, 1,, 39,, *1,,3 6 1,, 4,, 1 4,,

6 350 J. MELLANBY. A COMPARISON OF THE EFFECTS OF CALCIUM, BARIUM, STRONTIUM AND MAGNESIUM, CHLORIDES. No direct comparison of the efficiencies of pancreatic rennin and gastric rennin to coagulate milk can be made since there is no method of determining the absolute quantities of these ferments contained in any solution. But interesting results are obtained from a comparison of the effects produced by salts on their coagulating activities. Calcium chloride. The following is an example taken from a series of experiments. The milk was diluted with three times its volumne of water. The figures give the proportions of calcium salt and gastric rennin required to produce coagulation in approximately two minutes: Units of gastric rennin Amount of calcium salt in the milk 400 N N N N N 150 6N N From these figures it is evident that the dilution of the calcium content of milk below a minimal value (approximately 07 0/0 CaCl2) entails the addition of a comparatively large quiantity of gastric rennin to produce coagulation. A similar diminution in the quantity of rennin added does not demand a corresponding increase in the calcium salt to produce coagulation. Generally stated a large amount of gastric rennin is required to produce coagulation in calcium poor milk but a correspondingly large amount of calcium is not required to produce coagulation in milk to which a minimal amount of rennin has been added. Precisely similar facts were observed in the coagulation of milk by pancreatic rennin. The following figures are taken from a detailed series of experiments and show the quantities of calcium and pancreatic 150

7 RENNIN AND MfILK. rennin required to produce a coagulation time of approximately five minutes:- Units of pancreatic rennin Amount of calcium salt in the milk 100 N 150 These solutions did 10 2 N not coagulate N) 6 4N N Coagulation in five 150 minutes. 3 6N ~~~~~~21Nj 150 y The first two sets of figures above, taken from experiments in which the solutions did not coagulate, are included to illustrate the point that when the calcium content of the milk falls below a minimum value then no increase in the amount of pancreatic rennin added produces coagulation. A similar fact was shown to hold for gastric rennin. With pancreatic rennin the minimal calciuim content of the milk required for coagul]ation is approximately *11 e/o CaCl2. In coagulation by gastric rennin a fall in the calcium coutent of the milk below 07 e/o CaCI2 entails the addition of a large quantity of rennin; but even when the calcium content is reduced to 040/e CaCI2 coagulation is produced after the addition of a very large quantity of gastric rennin. Barium, strontium, calcium and magnesium chlo7ides. Milk was diluted with three times its volume of distilled water. This diluted milk did not coaguilate after the addition to it of gastric rennin or pancreatic rennin. In order to produce coagulation some salt containing a dibasic positive ion of the alkaline earth series was added to it. The comparative efficiencies of these salts were determined from a large series of experiments. The following table gives the amounts of pepsin solution required to produce coagulation in one minute of 3 c.c. of diluted milk containing *02N of each of these salts. Salt *02 N Glyc. of pepsin (1: 100) CaCL2 1 c.c. BaCl2 * SrCJ *35 MgC?2.5 All these salts are effective in producing the coagulation of -milk by gastric rennin. Calcium chloride is most effective, and magnesium

8 352 J. MELLANBY. chloride is least efficient whilst barium and strontium chlorides occupy intermediate positions. Precisely similar results were obtained from experiments on the coagulation of milk, containing these salts, by pancreatic rennin. The ability of these salts to replace one another in the rennin coagulation of milk is in marked contrast to the specific action of calcium in the coagulation of blood. Calcium salts are essential for the generation of fibrin ferment from prothrombin, and calcium cannot be replaced by salts of barium, strontium or magnesium. ADSORPTION OF RENNIN BY THE COAGLULUM AND BY EXTRAVENOUS PROTEINS. The following experiments were made to determine whether any evidence could be obtained of the union of caseinogen and rennin when coagulation occurred. A solution of caseinogen was made in the way described by Ringer(24). Milk, diluted with ten volumes of water, was precipitated by acetic acid, and the precipitated caseinogen after adequate washing was suspended in distilled water and ground up with powdered calcium carbonate. The milk white solution so obtained was placed in an ice chest for 24 hours. At the end of that time the excess of calcium carbonate had settled to the bottom of the flask and the fat which had been precipitated with the caseinogen had risen to the surface. This solution of caseinogen was used in the following experiments. Rennin was added to a solution of calcified caseinogen and the time of coagulation noted, thus: (A) Caseinogeu CaCJ2 *2 N H20 Rennin ('01 0/o) Coag. time 4 c.c. 1 c.c. 4 c.c. 1 c.c. 10 secs. The fluid (a) was expressed from the clot, and 5 c.c. of (a) were added to more calcified caseinogen solution and the time of coagulation noted. (a) Caseinogen CaCI2 2N H20 Coag. time 5 c.c. 4 c.c. *5 c.c. *5 c.c. 1 min. Similar experiments were made using varying quantities of ferment in the original tubes. The following figures give the coagulation times: Coag. time Coag. time A 1.c. rennin a (Expressed fluid 10 secs. 1 min. D.-4 c.c. rennin d Expressed fluid 20 secs. 2 mins. B 8 c.c. rennin 15 secs. E 2 c.c. rennin b texpressed fluid 1 m. e Expressed fluid 25 secs. 3j mins. C J6 c.c. rennin 15 Wecs. F ) 1 c.c. rennin c (Expressed fluid 1j mins f (Expressed fluid i min. 7j mins.

9 RENNIN A ND MILK. If Do rennin had been removed by the clot then the quantities of ferment in the tubes (a), (b), (c), (d), (e), (f), would have been *5 c.c., *4 c.c., *3 c.c., *2 c.c., 1 c.c., 05 c.c. Comparing, the times of coagulation of (a) with (F) it is evident that even in this case when 1 c.c. of rennin was added to the caseinogen there was less than 1 c.c. of ferment left in the residual fluid. The experiment gives conclusive evidence that in the coagulation of milk the rennin is adsorbed by the caseinogeni-the greater the quantity of rennin originally present the greater the quantity removed by the coagulum. In view of this fact the effect of finely divided coagulated egg-white or a solution of peptone was tried. The result was to prolong the coagulation time. The following figures show that Witte's peptone has a much greater inhibitory effect on the coagulation of milk than egg-white. Milk H20 CaCIh (I2N) Rennin Coag. time 2 c.c. 6 c.c. 1 c.c. 1 c.c. 1 min.,, + 1 grm. peptone No coag. in 4 hrs.,, + 1 grm. egg-white 3i mins. 353 This greater inhibitory effect of Witte's peptone might have been anticipated from the fact that the peptone goes into solution and so presents a much greater surface for the adsorption of the rennin. The inhibitory influence of coagulated egg-white on the coagulation of milk by rennin affords strong presumptive evidence that gastric rennin is identical with pepsin. THE PRESENCE OF ANTI-GASTRIC RENNIN AND ANTI-PANCREATIC RENNIN IN SERUM. The presence of anti-bodies and anti-ferments has long been recognised in serum. In view of this property of serum it was of interest to determine whether gastric rennin and pancreatic rennin were destroyed by it and if so whether the rates of destruction were the same in each case. A mixture of 1 c.c. gastric rennin, *2 c.c. ox serum and 8 c.c. water was made. After one hour -2 c.c. of the mixture was added to 2 c.c. of milk. Coagulation was not produced although a similar quantity of gastric rennin to which no serum had been added, coagulated the same quantity of milk in half a minute. It is evident, therefore, that serum contains a large quantity of some substance which destroys gastric rennin.

10 354 J. MELLANBY. A similar experiment was made with pancreatic rennin. Pancreatic rennin Serum H c.c. *0 0.c. A1,, *8,, *2,, A,, *~~~~6.,, 4 9" A3 After one hour 1 c.c. of each of A1, A2, A. was added to 2 c.c. of calcified milk. The following coagulation times were produced. Coag. time c. panoreatic rennin (Control) i min. 1,, AS 6 mins..1,, A2 8 1,, A1 15 From the above results it is clear that serum contains a substance which prevents pancreatic rennin from coagulating milk. But the amount of anti-pancreatic rennin present in serum is much less than that of anti-gastric rennin. The experiment affords conclusive evidence of the difference between gastric rennin and pancreatic rennin. THE ELECTRICAL STATE OF MILK DURING RENNIN COAGULATION. An investigation into the electrical state of milk during coagulation was demanded by the absolute dependence of the rennin coagulation on the presence of a minimal quantity of a divalent positive ion of the alkaline earths series in the milk. The determination of the electrical conductivity of milk during rennin coagulation was made by the ordinary galvanometer method for determining the conductivities of electrolytes. The electrodes used were carefully coated with platinum black before rmaking the determnitations. A mixture of milk and gastric rennin was put into the conductivity tube and the resistance determined after the lapse of varying intervals of time. The composition of the fluid in the tube, and its resistance after varying intervals of time was as follows:- Milk Mins. after H20 CaC12 (-2 N) Rennin adding rennin Resistance 5 c.c. 3-6 c.c. 1 c.c. *4 c.c ohms ,, 3 221,, 4 220,, 5 220,, 7i Coagulation took place in three and a half minutes in the control tube. From the above figures it may be seen that the resistance of the

11 RENNIN AND MILK. milk varied less than 05 0/0 between the tbird and fifth minutes, during which time coagulation took place. The results gave no evidence that during the rennin coagulation of milk the calcium enters into combination with the caseinogen so as to diminish its capacity for conducting an electric current. DISCUSSION OF RESULTS. The experimental work detailed in the previous pages affords an insight into two problems connected with the rennin coagulation of milk: the relation of rennin to proteolytic ferments, and the ultimate nature of rennin coagulation. (1) The relation of rennin to proteolytic ferments. The hypothesis is put forward that all proteolytic ferments coagulate milk provided the calcium content of the milk be adequate. Those, which like pepsin, act in an acidic mnedium, require the presence of a relatively small quantity of calcium salt in the milk; those, which, like trypsin, act in an alkaline medium, require the presence of a relatively large quantity of calcium salt in the milk, before producing coagulation. The ubiquitous distribution of the rennin enzyme and its association with living tissue whether animal or vegetable can be readily appreciated on the hypothesis that all proteolytic ferments coagulate milk. A comparison of the properties of gastric rennin and pancreatic rennin offers strong evidence in favour of the identity of rennin with proteolytic ferments. The differences between gastric rennin and pancreatic rennin are comparable to the differences between pepsin and trypsin. Gastric rennin is destroyed by alkali, pancreatic rennin is stable in alkaline solution; serum contains a large quantity of antigastric rennin, but only a comparatively small quantity of anti-pancreatic rennin; gastric rennin requires the presence of a much smaller amount of calciumn salt in milk to produce coagulation than pancreatic rennin. If it be postulated that the coagulation of milk is due to a special enzyme then it is necessary to assume that at least two different rennin enzymes exist. Probably the most conclusive evidence in favour of the hypothesis as to the identity of rennin with proteolytic ferments is obtained from a consideration of the generation of these proteolytic enzymes from their precursors. The work of Langley(X), and of Langley and Edkins(26) showed that just as pepsiinogen is converted into pepsin by the action of hydrochloric acid so this acid converts the precursor of gastric rennin into rennin. Also a neutral or alkaline extract of the gastric mucous membrane neither digests protein nor PH. XZLV

12 356 J. MELLANBY. coagulates milk, but the addition of hydrochloric acid to such an extract results in the immediate production of pepsin and rennin. Further the ease with which pepsin is destroyed by alkali and the comparative stability of pepsinogen in alkaline solution are paralleled by similar properties of rennin and its precursor in extracts of the gastric mucous membrane. Similar facts have been shown to hold true for trypsin and pancreatic rennin by Woolley and myself. A glycerine extract of a pancreas neither digests protein nor coagulates milk. Dilution with water or addition of enterokinase to such an extract results in the production of mnarked milk clotting and proteolytic activities. Further we have now worked out in detail the conditions under which pancreatic rennin is produced in fresh pancreatic juice. These results (which will be described in a subsequent paper) afford the strongest evidence that the coagulation of milk by activated pancreatic juice is due to trypsin and arguing from this conclusion it is logical to assume that the coagulation of milk by gastric rennin is due to pepsin. The identity of gastric rennin and pepsin has been discussed by numerous observers without any definite conclusion being reached. But in these discussions a number of important factors have not been fully considered. In any estimation of the comparative milk clotting and proteolytic activities of a solution it is necessary to note the very small quantity of rennin which coagulates diluted milk containing an optimum quantity of calcium chloride. In one experiment 000,002 grams of impure scale pepsin coagulated 2 c.c. of milk in three minutes; in a second experiment -1 c.c. of a thousand fold dilution of a glycerine extract of pepsin coagulated 3 c.c. of milk in four minutes. The solutions in the dilutions used did not digest fibrin or egg white, but in stronger concentrations showed all the properties of powerful proteolytic enzymes. Another factor which must be considered in these comparative experiments is the rate of destruction of proteolytic enzymes. A test for a rennin enzyme can be carried out in a few minutes and during this interval of time there is no appreciable destruction of it. But proteolytic determinations by means of congo red fibrin or Mett's tubes take hours for their completion and in these prolonged experiments factors involving the destruction of the proteolytic enzyme have ample opportunities to exercise their destructive effects. Trypsin in an alkaline medium deteriorates at a very great rate especially if there is no protein present in the extract. If a solution of trypsin free from protein be made alkaline to the extent of *5 0/0 Na2CO. and coagulated egg albumin be added to it the destruction of trypsin may be so

13 RENNIN AND MILK. 357 rapid that practically no digestion of the egg-white may be noted. If however a solution of egg albumin be used then well marked proteolysis can be noted owing to the fact that the trypsin is protected against destructive agents by the protein dissolved in the medium. -But although protein in solution protects proteolytic enzymes against the destructive action of other agencies it is important to recognise that -proteins mask the milk clotting properties of rennin solutions. The addition of a solution of protein to a rennin extract diminishes the milk clotting property of the extract; and it is possible to prepare a solution in which proteolytic properties can be demonstrated by means of Mett's tubes in which rennin effects can be detected with difficulty. On the addition of protein to a rennin solution the ferment divides itself -between the added protein and the caseinogen in proportion to their relative avidities for it and depending on this relation the resultant milk clotting activity of the rennin solution is determined. Finally arguments have been brought forward against the hypothesis that gastric rennin is identical with pepsin insomuch that solutions which contain rennin are actively antipeptic and vice versa. These arguments depend upon the non-recognition of the fact that it is possible for one substance to produce two entirely different effects under different conditions and that these conditions may be inimical to one another. To take an extreme case-suppose a solution of pepsin contained *2 0/o potassium oxalate. This solution even though gastric rennin were identical with pepsin would show no milk clotting action and moreover would be actively anti-rennetic to any rennin solution which might be added to it. A similar inhibitory effect on milk coagulation is produced by sodium chloride in larger quantities. Again take the case of an active milk clotting extract of the pancreas. The rennin activity of such an extract is largely augmented by the addition to it of a calcium salt and yet the presence of such a salt inhibits the action of the trypsin contained in it, owing to the calcium diminishing the effective alkalinity of any solution to which it may be added. (2) The nature of the processes involved in the coagulation of milk. Certain fundamental facts may be observed in the coagulation of milk bv rennin. These are:- (a) The inverse proportionality which exists between (i) the rennin and coagulation time when the caseinogen and calcium are kept constant; (ii) the caseinogen and coagulation time when the rennin and calcium are kept constant; (iii) the calcium and coagulation time when the rennin and caseinogen are kept constant. 24-2

14 358 J. MELLANBY. (b) The loss of this proportionality when (i) the rennin falls below a certain minimum value; (ii) the calcium falls below a certain minimum value; (iii) the caseinogen rises above a certain maximum.value, the minima and maxima in the above being functions of the relative quantities present of the other variables involved in the coagulation process. (c) The independence of the coagulation time on the volume of the fluid in which the caseinogen and rennin are contained always provided that the percentage of calcium salt is kept constant. (d) The removal of a large quantity of rennin by the clot produced in the coagulation process. These experimental results may be explained on the hypothesis that in the coagulation of milk the rennin forms an adsorption compound with the caseinogen and this adsorption compound is precipitated by the divalent calcium ion. The first process involves a distinct time factor; the second process is practically instantaneous. Ionised calcium salts have a marked effect on the state of aggregation of the caseinogen contained in milk as was shown by Ringer(24) in numerous investigations. The addition of calcium chloride increases the whiteness of milk although it does not prodtice precipitation. Again fresh milk can be boiled without producing coagulation of the caseinogen but milk to which calcium chloride has been added coagulates at. varying temperatures depending upon the quantity of added calcium salt. But in order to produce coagulation (apart from acid coagulation) some rennin must be present. Calcium ions alone cannot precipitate caseinogen yet this same caseinogen weighted with adsorbed proteolytic ferment can be so precipitated, and the quantity of ionised calcium salt required to effect such precipitation is intimately related to the quantity of ferment adsorbed by the caseinogen. The hypothesis may be represented as follows: (i) (x) caseinogen + (y) proteolytic ferment-...{(x) caseinogen (z) ferment} + (y -2) ferment. (ii) in presence {(x) caseinogen (z) ferment} -- coagulum. of CaCl2 (a 0/0) With a definite calcium content of the milk it is evident that the formation of the complex {(x) caseinogen (z) ferment} takes place in times proportional to the relative quantities of caseinogen and ferment present provided there is sufficient ferment to combine with the caseinogen to the requisite degree of saturation, a degree which is in turn dependent upon the quantity of calcium salt contained in the solution.

15 RENNIN AND MILK. 359 If however the ferment added be less than sufficient to combine with the caseinogen to form the complex {(x) caseinogen (z) ferment} then a portion of the caseinogen must first be digested by the proteolytic ferment and only after a varying time does the proportion of ferment to caseinogen become as (x) is to (z) and coagulation occur. The sudden loss of inverse proportionality in the curve expressing the relation of the coagulation time to the amount of ferment added corresponds to a point where the proteolytic ferment is present in too small a quantity to saturate the caseinogen and the delayed time in coagulation is due to the time required to digest the excess of caseinogen. The relation of (x) to (z), of caseinogen to ferment in the coagulation process, is determined by the calcium content of the fluid-if the calcium content be optimal then the required proportion of ferment to caseinogen is small; if however the calcium content of the milk be minimal the proportion of ferment to caseinogen must be large before coagulation occurs. But however much ferment may be added some divalent positive ion must be present before coagulation occurs; and similarly however much calcium may be added some ferment must be present before coagulation occurs. In the case of pancreatic juice if no calcium salt be added to milk trypsin digests the caseinogen and does not form a coagulum.. This dependence of the comparative amounts of caseinogen and proteolytic ferment in the protein-ferment complex on the calcium content of the mnilk affords an explanation of the similarity of the effects observed on the coagulation time when the calcium or rennin content of the milk is increased. An increase in the amount of calcium present in the milk necessitates that fewer ferment groups should combine with the caseinogen before precipitation occurs. The calcium salt probably exerts its precipitating action by virtue of the electrical charge on the divalent calcium ion. This hypothesis is supported by two observations: (a) That the calcium salt can be replaced by any salt of the alkaline earths containing a divalent positive ion (the salts of barium, strontium or magnesium) and (b) That no change takes place in the electrical conductivity of the milk during coagulation. The question arises as to the difference between caseinogen and casein, i.e. the difference between the protein in the milk and the precipitated protein in the clot. When first formed the clot consists of an adsorption complex of caseinogen and proteolytic ferment. Even

16 360 6 J. MELLANBY. if the activity of th-e ferment were destroyed immediately after the production of coagulation it is doubtful whether the ferment nucleus would be split off from the caseinogen so that the original caseinogen might be recovered. But in any experiment some ferment must be adsorbed by the caseinogen for an appreciable time before precipitation occurs and during this time it is probable that the process of digestion is initiated and the clot when formed differs by this degree of digestion from the original caseinogen. The least change takes place when coagulation is produced by a minimal amount of rennin in the presence of an optimal amount of calcium. How slight these digestive changes may be is evident from the controversy on the differences of caseinogen and casein. These two substances possess identical physical and chemical properties with one exception only-a caseinogen solution is coagulated by rennin and calcium salts, a solution of casein is not coagulated by this procedure. The comparative results obtained from experiments on gastric rennin and pancreatic rennin throw some light on the controversy whether when pepsin acts in an acid medium the pepsin combines with the hydrochloric acid; and similarly for trypsin and sodium carbonate. The coagulation of milk by pepsin requires the presence of much less calcium salt than that produced by trypsin; also acids assist the coagulation of milk and alkalies depress it. These two facts indicate that pepsin may be a complex containing hydrochloric acid and trypsin a complex containing sodium carbonate, the adjuvant effect of the acid and the depressant effect of the alkali being responsible for the greater quantity of calcium required by trypsin in comparison to pepsin to produce coagulation. But although pepsin may be a hydrochloride of pepsinogen there is no indication from the experiments of Woolley and myself that trypsin is a sodium carbonate compound of trypsinogen. The more ready hydrolysis of proteins to amino acids by trypsin suggests that this ferment is a more effective proteolytic agent than pepsin and the smaller size of the trypsin-caseinogen aggregates would be in accordance with the smaller number of trypsin molecules required to hydrolyse the caseinogen. Again experiments on milk coagulation offer a ready method for the detection and estimation of proteolytic ferments. Milk diluted with four times its volume of *1 0/ OaCl2 is coagulated by small quantities of pepsin or trypsin. In quantitative experiments it is advisable to guard against precipitation of the caseinogen of the milk by acid contained in the solution to be tested. This can be accom-

17 RENNIN AND' MILK. 961 plished by adding a little powdered calcium carbofiate to the ferment solution until it gives a neutral reaction to litmus. Further it is important to remember that large quantities of salt inhibit the coagulation' of milk and that the rennin properties of proteolytic ferment solutions are masked by dissolved protein. These difficulties may be overcome by the use of glycerine for the purposes of extraction. Glycerine readily dissolves ferments and the solutions so obtained retain their activities for considerable periods of time, whilst very little protein is contained in them. With short coagulation times it may be assumed that the amount of ferment contained in the solution is inversely proportional to the time of coagulation,' but in a series of experiments it is advisable to prepare a standard curve by taking a known solution of the ferment and determining the times of coagulation of milk after adding varying quantities of ferment under defined conditions. The quantity of ferment contained in an unknown solution can be ascertained by seeing what quantity of ferment corresponds to the same coagulation time on the standard curve obtained under conditions similar to those used in the determination of the standard curve. SUMMARY. (1) The coagulation of milk by pancreatic rennin follows the same general laws as coagulation by gastric rennin. (2) Pancreatic rennin is a ferment entirely different and distinct from gastric rennin as is shown by (a) the generation of these two ferments from their precursors in the gastric mucous membrane and the pancreas respectively; (b) the action of alkali upon them; (c) the different quantities of anti-gastric rennin and the antipancreatic rennin in serum; (d) the greater quantity of calcium salt required in milk to produce coagulation by pancreatic rennin. (3) In the coagulation of milk by pancreatic rennin or gastric rennin the calcium salts may be replaced by salts of barium, strontium or magnesium. (4) There is no indication from electrical conductivity determinations that calcium enters into, chemical combination during rennin coagulation. (5) The hypothesis is put forward that all proteolytic ferments coagulate 'milk provided suitable conditions be provided for their

18 362 J. MELLANBY. action; those which, like pepsin, act in an acidic medium, require the presence of a relatively smaller amount of calcium mn the milk to produce coagulation than those which, like trypsin, act in an alkaline medium. In order to produce typical rennin coagulation in milk by trypsin it is necessary to increase the normal calcium content of milk. (6) On this hypothesis the coagulation of milk is due to the adsorption of the proteolytic ferment by the caseinogen and the precipitation of this ferment-caseinogen complex by the divalent calcium ions present in the milk. Further the quantity of ionised calcium salt required to effect precipitation is intimately related to the quantity of ferment adsorbed by the caseinogen. (7) A method based on this hypothesis is described for the detection and estimation of proteolytic ferments. (The expenses of this Research have been defrayed by a grant from the Government Grant Committee of the Royal Society.) REFERENCES. (1) Hammarsten. Maly'sJhrsb. ii. (2) Pavlov. Verhandl. d. Gesellsch. russ. Aerzte. (3) Pavlov and Parastuschuk. Ztschr. physiol. Chem. XLII. p (4) Nencki and Sieber. Ibid. xxxii. p (5) Winogradow. Pfluiger's Arch. LXXXVII. p (6) Gewixi. Ztschr. physiol. Chem. LIV. p (7) S a w j al o f f. Ibid. XLVI. p (8) Jacoby. Biochem. Ztschr. i. p (9) van Dam. Ztschr. physiol. Chem. LXIV. p (10) Funk and Niemann. Ibid. LXVIII. p (11) Michaelis. Biochem. Ztschr. xvii. p (12) Hammarsten. Ztschr. physiol. Chem. LXI. p ; LXVIII. p (13) Rakoczy. Ibid. LXVIII. p ; LXXII. p (14) Taylor. Journ. Biol. Chem. v. p (15) van Hasselt. Ztschr. physiol. Chem. LXX. p (16) Porter. This Journal, XLl. p (17) Roberts. Proc. Roy. Soc. xxxii. p (18) Edkins. This Journal, xii. p (19) Brodie and Halliburton. Ibid. xx. p (20) Vernon. Ibid. xxvii. p (21) Mellan by and Woolley. Proc. Physiol. Soc. p. xi, (This Journ. xxxix.) (22) Fuld. Btrg. chem. Physiol. u. Path. ii. p (23) Reichel and Spiro. Ibid. vii. p (24) Ringer. This Journal, xi. p (25) Langley. Ibid. iii. p (26) Langley and Edkins. Ibid. vii. p