THE MILK-CLOTTING ACTION OF PAPAIN*

THE MILK-CLOTTING ACTION OF PAPAIN* BY A. K. BALLS.4ND SAM R. HOOVER (From the Food Research Division, Bureau of Chemistry and Soils, United States Department of Agriculture, Washington) (Received for publication, August 18, 1937) Although the milk-clotting action of papain has been recognized for many years, this property of the enzyme has received little quantitative attention. It appears to be, however, a characteristic property of the proteolytic component of this enzyme system. Various workers have brought forth evidence as to the composite nature of the papain system, which has at least two components. In the most recent work Bergmann and coworkers (l-3) have tentatively designated them as Papain Peptidases I and II. The first one is characterized as splitting benzoylisoglutamine and hippurylamide, and is completely inhibited by phenylhydrazine and other carbonyl reagents. The second splits peptone ex a&mine and gelatin, and is activated by phenylhydrazine. Controlled oxidation with iodine inactivates both components; subsequent reduction with HCN restores only the activity of Peptidase II. Method A solution of dried whole milk was found by Kunitz (4) to be a more reproducible substrate for clotting experiments than fresh milk. 20 gm. of dried milk were ground to a smooth paste with a small amount of diluted acetate buffer, ph 4.60, and diluted with this buffer to 100 cc. The liquid was then filtered through cheese- * Food Research Division Contribution No. 339. 1 A standard buffer is prepared by mixing 2 volumes of M CH&IJOOH with 1 volume of M NaOH. 10 cc. of this buffer diluted to 85 cc. and added to the dried milk give a total volume of 100 cc. The concentrated buffer cannot be added directly to the dried milk or first paste without affecting the stability of the emulsion obtained. 737

738 Milk-Clotting Action of Papain cloth. It is stable for several weeks when kept in the ice box under toluene. The test solutions of papain were prepared by grinding the crude enzyme to a smooth paste with a small amount of water in a mortar. The paste was diluted (with grinding) to the volume required2 and then filtered through paper. 1 cc. of various dilutions of this solution was incubated in test-tubes 15 mm. in diameter at 40 with 10 cc. of the milk preparation. The time was observed when the tubes showed a thickening on tilting; this is prior to the complete setting and is rather sharp. In the case of papain, the time of clotting is apparently unaffected by this shaking. An excess of activator appears to be required in order to obtain complete activation. With all preparations except fresh latices, it is therefore necessary to activate the enzyme first. This was done by diluting with H2S water and bubbling H&S through the solution for 30 to 60 minutes at room temperature. Treatment at 40 with an excess of cyanide or cysteine (both at ph 5) was also satisfactory. Natural papain can be tested in freshly prepared solutions on freshly prepared milk. A short clotting time must also be used to avoid oxidation. For practical purposes it is best to choose an amount of enzyme that can be expected to clot the milk in from 1 to 5 minutes and to observe the time with a stop-watch. A glass-walled thermostat permits the tube to be tilted without removal from the bath. Experiments on the same enzyme solution show that the value for the clotting time can if desired be reproduced with great accuracy. Duplicate results do not ordinarily vary by more than ~5 per cent. Differences in activation of the enzyme may easily lead to larger deviations, however. Kinetics of the Clotting Process-Except for very small concentrations of enzyme, the time required for clotting is inversely proportional to the amount of enzyme present. The relation between clotting time and enzyme concentration is therefore a * A 5 to 10 per cent solution (based on the weight of dry enzyme used) is recommended. We have repeatedly observed that dilute papain solutions without added activator lose activity on standing, to some extent irreversibly. See also (5).

A. K. Balls and S. R. Hoover 739 straight line, as shown in Fig. 1. Where E is the weight of enzyme (in mg.), and t the time (in minutes), E = K/t (1) It follows from Equation 1 that E = K when t = 1; therefore, the activity per mg. (l/e) is l/k. For all purposes connected FIG. 1. Relationship between clotting time (reciprocal) and amount of enzyme. Curves I, II, III, and IV represent HzS-activated papains; Curve V, Natural Papain I. with the assay of papain preparations, this expression is sufficiently accurate. At low concentrations of enzyme, however, the relationship no longer holds. The time required for coagulation by a small dose

740 Milk-Clotting Action of Papain of enzyme is much longer than would be expected from the foregoing, and the system behaves as though a part of the enzyme did not take part in the clotting reaction. Thus the curves of Fig. 1 do not pass through the origin (as they do in milk coagulation produced by chymotrypsin) but instead cut the axis of enzyme quantity at a finite value. Below this value the enzyme, though present, does not clot in any reasonable time. Experiments have shown, however, that amounts of enzyme too small to clot the milk within a reasonable time nevertheless have some effect on the system, because such pretreatment of the milk reduces the clotting time observed when an adequate quantity of enzyme is subsequently added. If c denotes the amount of enzyme removed from action during the clotting,3 then the available enzyme is E - c, and the previous expression (Equation 1) becomes (E - c)t = K (2) This accurately describes the relation between time and enzyme concentration over the range of our experiments. The magnitude of the deviation from direct proportionality is shown in Table I for Specimen IV of Fig. 1. The quantity of enzyme inactive during the clotting may be measured thus for any particular papain preparation, and expressed in terms of weight. (In Specimen IV, Table I, this quantity is 0.29 mg.) If, however, the activity of the preparation is taken into account, it may be seen that the same amount of enzyme activity is lost to the clotting reaction in all cases, independent of the preparation used. This is shown in Table II, where the weight of enzyme inhibited (c) is multiplied by the unit activity (l/k). The product c/k equals the inhibition per unit activity. In Table II c and K are determined by inspection of Fig. 1, and c/k is shown to be substantially constant for all the samples of papain studied. Evidently the milk contains a substance capable of inhibiting the active enzyme. The amount of enzymic activity inhibited is a property, not of the enzyme used, but of the substrate. J c is determined either by solving simultaneous equations for the various points or by determining the intercept on the E axis when l/t is plotted against E.

A. K. Balls and S. R. Hoover 741 The kinetics of milk clotting by papain, therefore, vary from those observed for rennin by Holter (6) and for chymotrypsin. The clotting with rennin is represented by the expression E(t - z) = K, where x is probably the time lag of clotting after proteolysis has TABLE Amount of Enzyme (Specimen IV) versus Clotting Time I Amoux~; 11; aym Time of coagulation I#.:= K EC -2; ;,K c. w. min. 2.0 2.25 4.5 3.9 1.0 5.25 5.25 3.7 0.75 8.33 6.25 3.8 0.50 18.0 9.0 3.8 TABLE Showing Loss in Enzymic Activity Is Independent of the Preparation Used II Papain c K C/K -- HZS-Activated Specimen I................. 0.23 2.55 0.090 I I( II........... 0.32 4.85 0.066 Natural Specimen I.................. 3.0 48 0.063 Hkl-Activated Specimen III............ 0.27 4.0 0.067 I I IV................. 0.29 4.1 0.071 Temperature C. 60 49 36.6 30 _- - - TABLE Effect of Temperature on Clotting Time Time of clotting E.l = K I T; Ibmperature 5 mg. 2.5 mg. 6 mg. 2.5 mg. min. min. 2.5 5.0 4.5 8.8 14.0 28.5 32.0 67.0 III 12.5 12.5 22.5 22.0 70.0 70.6 160.0 168.- - - C. coefficient 1 &IO 50-60 1.7 40-50 2.4 30-40 3.4 reached the requisite stage. In chymotryptic clotting, neither the rennin nor the papain equation fits, but the simple relationship E. t = K holds over a 30-fold dilution range. Fig. 2 contains data of $4. Schwimmer, of this laboratory, for crystalline chymo-

742 Milk-Clotting Action of Papain trypsin on the milk preparation previously described.4 Apparently there is a fundamental difference in the reactions of milk clotting by these three enzymes. Efect of Temperature on Clotting Time-The temperature coeflicient of the clotting time was determined with unactivated papain in a sufficiently high concentration for c to be negligible in Equa- 0 CHYMOTRYPTIC CLOTTING OF MILK AT 30.02 34.06.08 JO J2 J4 J6.I8 Emg* FIG. 2. Relationship between clotting time (reciprocal) and amount of chymotrypsin. The asterisk indicates mg. of dried filter cake, containing MgS04. tion 2. The values for Qlo shown in Table III were taken from the smooth curve of the activity-temperature relationship. It is evident that Equation 1 holds at lower temperatures for much longer time intervals than at 50. 4 In order to avoid the possible influence of agitation during chymotryptic clotting, the recorded clotting time was determined after several preliminary runs for the purpose of approximating it.

A. K. Balls and S. R. Hoover 743 Activation and Effect of Phenylhydrazine-Activation of the clotting power by cysteine and H&3 is complete in 15 minutes, and the activated enzyme is remarkably stable. In the presence of reducing substances such as cysteine or cyanide, solutions have been kept at 40 for days with scarcely detectable change in potency. There is no significant change in this property whether the phenylhydrazine is added with or after the activator. Table IV shows the effect of phenylhydrazine on clotting time. Neutralized phenylhydrazine hydrochloride was incubated with the enzyme at 40 for 15 minutes. Natural papain, on the other hand, is clearly activated by 0.05 M phenylhydrazine. This is presumably due to the reduction of the TABLE Effect of Phenylhydrazine on Clotting Time Enzyme Natural papain...... I I....t... I................... H&3-activated papain........... I............. < l.............. IV Amount mg. 10.0 10.0 2.5 2.5 2.5 2.5 Time of clotting Y min. 0 4.5 0.05 0.67 0.05 2.5 0 1.75 0.5 1.67 0.25 1.67 natural activator by the reagent, analogous to its known reduction of methylene blue.5 Assay of Milk-Clotting Activity of Papain-By working with sufficient enzyme, it is possible to ignore the amount of ferment destroyed during the reaction, and so to use the expression E = K/t (Equation 1). If a unit of activity is defined as the amount of enzyme which clots the milk in one test-tube (under the conditions described) in 1 minute, then the units of enzyme present in E mg. of preparation are l/t, and units of enzyme per mg. of preparation used = l/e-t. This method of assay shows quite satisfactory agreement with the casein digestion method 6 Bergmann and Ross (2) advance another explanation of the activation by phenylhydrazine as due to a shift in a dissociation equilibrium of a postulated compound of the two peptidases.

744 Milk-Clotting Action of Papain previously worked out in this laboratory (7) and also with the proteinase activity as shown by the determination of nitrogen not precipitable with trichloroacetic acid, after the manner used by Anson (5), but with casein as a substrate (Table V). It is not to be expected that the agreement between clotting units and titration units will hold in all cases, because on the same enzyme preparation, the casein-splitting activity may be reduced by the addition of phenylhydrazine, without affecting the milkclotting activity. It is evident, however, that for normal ferment TABLE Assay of Papain by Various Methods Papain V Clotting units H&-Activated Specimen I... 0.39 I I II.... 0.21 I I III... 0.25 I, IV.... 0.24 Fresh Latex I... 1.00 Titration unite* 0.20 0.11 0.12 0.11 Protein 43 27 31 35 142 * The titration units used are the reciprocal of the K obtained according to the method of Balls, Swenson, and Stuart; this is done to obtain both figures as activity per mg. t 2 per cent casein was digested at ph 5.6 with KCN-papain and the nitrogen rendered non-precipitable with trichloroacetic acid was determined by Anson s (5) method. The figures are purely arbitrary units and refer to tyrosine equivalents liberated at 30 per mg. of enzyme, averaging 3 minute and 5 minute digestion intervals. (The function is practically linear for short time intervals.) preparations and for short periods of digestion, the titration values will, as a rule, be comparable to the milk-clotting assay. The increase in nitrogen not precipitable by trichloroacetic acid seems, however, to be the nearest approach to true proteinase action, and the values obtained for this activity over a 5-fold range are in good agreement with the relative power of these specimens of papain to clot milk. There is every indication that the milk-clotting power of papain represents its proteinase activity. Nt SUMMARY On the basis of the experiments recounted here, the milk-clotting component of papain appears to agree rather closely with the

A. K. Balls and S. R. Hoover 745 current conception of a papain proteinase. It is activated by H&3, cysteine, and cyanide, has a high temperature optimum, and is not inhibited by phenylhydrazine but rather is activated by this substance. A study of the kinetics of the clotting effect showed that the time required is a straight line function of the enzyme concentration, as is the case with chymotrypsin, but with this difference, that a quantity of the enzyme-constant for any given preparation-is inhibited by the milk. The relationship is accurately expressed by an equation of the form (E - c)t = K, as explained in the text. The rapidity and simplicity of the technique, and the probable connection between the clotting time and the proteolytic properties of papain, make the determination of the clotting power a valuable aid in describing the activity of this mixture of enzymes. BIBLIOGRAPHY 1. Bergmann, M., and Ross, W. F., J. Biol. Chem., 111, 659 (1935). 2. Bergmann, M., and Ross, W. F., J. Biol. Chem., 114, 717 (1936). 3. Bergmann, M., and Zervas, L., J. Biol. Chem., 114, 711 (1936). 4. Kunitz, M., J. Gen. Physiol., 18, 459 (1935). 5. Anson, M. L., J. Gen. Physiol., 20, 561 (1937). 6. Holter, H., Biochem. Z., 266, 160 (1932). 7. Balls, A. K., Swenson, T. L., and Stuart, L. S., J. Assn. Of. Agric. Chem., 18, 140 (1935).

THE MILK-CLOTTING ACTION OF PAPAIN A. K. Balls and Sam R. Hoover J. Biol. Chem. 1937, 121:737-745. Access the most updated version of this article at http://www.jbc.org/content/121/2/737.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/121/2/737.citation.full.h tml#ref-list-1