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Activation of Prorennin by Pepsin1, 2
Activation of Prorennin by Pepsin 1, A. G. RAND 8 and ¢. A. ERNSTROM'
Deprtment of Food Science and Industries, University of Wisconsin, Madison Abstract
The ability of pepsin to catalyze the activation of prorennin was demonstrated when purified prorennin solutions buffered at p H 6.0 and 5.5 were activated by pepsin at 25 C with little interference from autocatalytic activation by rennin. Addition of ~ NaC1 retarded activation of prorennin by pepsin at p H 6.0 and 5.5, but the reaction was still faster than in rennincatalyzed activation mixtures containing the same concentration of salt. Pepsincatalyzed activation of prorennin at p H 6.0 appeared to follow zero-order kinetics during the early stages of reaction, followed by a marked change to a reduced rate of activation. However, the kinetics of activation may have been complicated by lack of pepsin stability at p H 6.0. Pepsin was more proteolytic than rennin on casein substrates at all p H values from 2.0 to 6.0. The general proteolytic activities of rennin and pepsin as distinguished from their milk-clotting activities may be related to their respective abilities to activate prorennin. Pepsin was able to stimulate activation of crude rennet extracts at p H 5.5, where rennin has good stability. Use of pepsin in commercial rennet activation could reduce or eliminate activation losses. The common proteolytlc enzymes found in the digestive tract of animals are secreted as inactive precursors. Activation is usually accomplished by a proteolytic splitting of the z3~nogens into active enzymes and peptide fragments. Proreunin is the predominant enzymatic secretion of the abomasum of young calves, and in the acid environment of the stomach is rapReceived for publication June 12, 1967. 1 Published with approval of the Director, Wisconsin Agricultural Experiment Station. This study was supported in part by a research grant from the Research Committee of the Graduate School, University of Wisconsin, from funds supplied by the Wisconsin Alumni Research Foundation. 3Present address: Department of Animal Science, University of Rhode Island, Kingston. "Present address: Department of Food Science and Industries, Utah State University, Logan.
idly converted into rennin. The activation reaction is mainly autocatalytic, in which rennin attacks prorennin to produce more rennin (20). Above p H 6.0, activation is insignificant. On the other hand, rennin has excellent milkclotting activity at p H 6.7. Literature coneerning rennin action on casein suggests that the enzyme may function differently at different p H values. Between p H 3.0 and 4.0, it has maximum general proteolytic activity, and gives rise to noncasein nitrogen compounds which contain aromatic amino acids (2, 4, 7). The milk-clotting activity of rennin above p H 6.0 is attributed to its ability to liberate a glycomacropeptide from casein. Under these conditions, hydrolysis is rather specific and the liberated peptides contain few aromatic amino acids capable of absorbing ultraviolet light (13). This suggests that the ability of rennin to catalyze the activation of prorennin might be associated with its general proteolytic function and not with its specialized milk-clotting activity. I f this is true, other general proteolyric enzymes might also catalyze the activation of prorennin. As a calf grows, and consumes i n c r e a s i n g quantities of solid feed, pepsin gradually replaces rennin as the main proteolytic enzyme of the stomach. Consequently, it is not surprising that pepsin is found in some commercial renent extracts (16) ; and it is a matter of interest to know whether and under what conditions it will catalyze the activation of prorennin. Zymogens such as pepsinogen and trypsinogen normally activated by autocatalysis (10, 15) can also be activated by other proteolytic enzymes (8, 14, 19). Activation of chymotrypsinogen can be catalyzed by trypsin (19) or by a protease from Bacillus subtilis (1). Ege and Lundsteen (5) reported that pancreatin would activate prorennin in rennet extracts above p I I 5.5. Linklater (16) used pepsin to increase the activation rate of purified prorennin at p H 4.7 in solutions containing 15% sodium chloride. However, it is known that considerable autocatalytic activation of prorennin can also occur under these conditions (20). Conclusive evidence of the ability of pepsin to activate prorennin would require conditions under which autoeatalytic activation by rennin could be blocked or markedly inhibited without producing a similar effect on the action of pep-
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PRORENNIN ACTIVATION sin. The purpose of this study was to find such conditions, and to investigate the characteristics of the pepsin-catalyzed activation reaction. Experimental Methods
1757
filter paper had been washed in several changes of distilled water to remove residues capable of absorbing light at 280 m~. TCA filtrates were analyzed for nonprotein nitrogen (NPN) and for absorbancy at 280 m~. Blanks were prepared at each p H for both enzymes by adding 5 ml of buffered 3% casein solution to 1 ml of 72% TCA, followed by 0.1 ml of the enzyme solution. A f t e r 10 rain the precipitate was removed by filtration, and blank values for N P N and absorbaney determined on the filtrate. Activation of prorennin. Solutions of pepsin and rennin were tested for milk-clotting activity, and diluted so that 1 ml would give the desired activity in the final activation mixture. Activation mixtures were prepared by adding ] ml of diluted enzyme solutions to 15 ml of 0.3 ~ sodium citrate buffer at p H 5.5 or 6.0, followed by the addition of 2 ml of a concentrated prorennin solution. I n control samples the enzyme was replaced by an equivalent volume of distilled water. Samples were removed at regular intervals and tested for milkclotting activity.
Enzyme activity. Milk-dotting activities of both rennin and pepsin were determined as previously described (6). The definition of a unit of milk-clotting activity rennin unit (RU) was also reported earlier (20). The same definition was applied to the milk-clotting activity of pepsin. Nitrogen analysis. Nitrogen was measured by a semimicro-Kjeldahl procedure (11). Enzymes. Crystalline rennin was prepared by a method already described (6). Three-timescrystallized pepsin was obtained from Pentex Inc., Kankakee, Illinois. Purified prorennin was isolated from ealf-stomach tissue by a salt fractionation and D E A E chromatographic procedure (20). Casein digestion. Acid casein was prepared according to the method of Van Slyke and Baker (22), and dried with acetone. Two 6% stock solutions of casein were prepared, one Results A comparison of the hydrolytic actions of at p H 6.0 and another at p H 2.5. Separate portions of dry casein were mixed with small rennin and pepsin in casein substrates between amounts of distilled water, placed in an ice p i t 2.0 and 7.0 is shown in Fig. 1. Results are bath, and constantly agitated with a magnetic plotted in terms of nonprotein nitrogen and stirrer. Sodium hydroxide (0.1 ~ ) was added absorbaney at 280 in~ in 12% TCA filtrates. Above p H 5.5, rennin liberated nonprotein to one chilled suspension and agitated until the casein was dissolved at p i t 6.0. The second was nitrogen from casein, but very little of this similarly adjusted to p H 2.5 with 0.1 ~ HC1, material absorbed light at 280 m~. TCA filand agitated until solution was achieved. Each trates from samples treated with pepsin at p H solution was diluted to the required volume with 5.5 and 6.0 not only contained more nonprotein distilled water. Aliquots of the stock solutions nitrogen than those treated with rennin, but were then diluted with equal quantities of 0.3 M buffer to give final casein concentrations of Z © 3%. Buffers used were p H 2.0, phosphate; p H - - absorbancy Z ~'~ --nonprotein n;trogen 3.0, citrate; p H 4.0, lactate; p H 5.0, 5.5, and -U "~%~ ~ rennin pepsin 6.0, citrate; p H 6.5 and piE[ 7.0, phosphate. 2.0 0.2 0 -4 The stock casein solution at p i t 2.5 was mixed o rq Cq with buffers from p H 2.0 to 4.0; the stock z solution at p H 6.0 was mixed with the buffers / ~ \\ \ -4 >from p H 5.0 to 7.0. o / \ \ Solutions of crystalline rennin and pepsin 0 10 o.1 orq were prepared so that the addition of 0.1 ml z of enzyme to 5 ml of a buffered 3% casein frsolution would give a final activity equivalent O to 1 RU per milliliter in the reaction mixtures. The enzymes were allowed to act for I hr 0 0 at 25 C. The reaction was stopped by the 2.0 3.0 4.0 5.0 5,0 ZO addition of sufficient 72% trichloroacetie acid pH (TCA) to give a final concentration of 12% FIG. 1. Effect of p i t on nonprotein nitrogen in the reaction mixture. Ten minutes after the and absorbancy (280 m~) of 12% trieh]oroacetic reaction was stopped, the mixture was filtered acid filtrates following digestion of casein with through no. 42 Whatman filter paper. The pepsin and rennin for 1 hr at 25 C. J . D A I R Y S C I E N C E , VOL. 51, NO. 1 1
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they also had higher absorbancy values. The proteolytic optimum for the action of rennin on casein appeared to lie near p H 4.0, as previously reported by Foltmann (7). The proteolytic activity of pepsin increased as the p H was reduced to 2.0. Even though its optimum p H for the digestion of casein was well below that of rennin, pepsin still exhibited greater general proteolytie activity than rennin at p H 5.5 and 6.0. Activation of prorennin by pepsin. Activation mixtures containing prorennin at p H 6.0 were treated with two and four milk-clotting units of pepsin, respectively. Other samples were treated with zero and two milk-clotting units (RU) of rennin for comparison. Activation curves for each sample are shown in Fig. 2. A rapid initial increase in activity followed by transition to a more gradual rate of activation occurred in the prorennin samples containing added pepsin. More than 90% of the potential reninn was activated in 360 hr, while autocatalytic activation resulted in the activation of only 20% of the potential rennin in about 300 hr. Addition of rennin had little effect on rate of activation. The slightly higher activity was owing to added rennin. Although the experiment at p H 6.0 clearly demonstrated that pepsin could catalyze the activation of prorennin, rate of activation was extremely slow. Therefore, a similar experiment was conducted at p H 5.5. Results are presented in Fig. 3. Activation of samples containing added pepsin proceeded rapidly at first, then continued more slowly until maximum activity was reached after about 50 hr. Autocatalytic activation by rennin, although somewhat faster than at p H 6.0, resulted in only 45% of maximum activity in 180 hr and did not bring about complete activation even after 360 hr. Rennin had a greater catalytic effect at p H 5.5 than at p H 6.0, as demonstrated by the slightly higher rate of autocatalytic activation.
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Fie. 3. Pepsin- and rennin-catalyzed activation of prorennin at pH 5.5 in 0.3 ~ sodium citrate buffer at 25 C. Addition of 1~ NaC1 to activation mixtures retarded pepsin-catalyzed activation of prorennin at both p H 6.0 and 5.5. However, it was still considerably faster than in rennin-catalyzed controls containing the same concentration of salt.
Activation of crude rennet extracts by pepsin. Unactivated commercial extract s at 2 C was divided into several 300-ml portions and each one adjusted to the desired p H with 1 N lactic acid or disodium phosphate. One milliliter of pepsin solution was then added to 20 ml of the cold extract. Distilled water was substituted for pepsin in control samples. Pepsin solutions were prepared to provide the desired milkclotting activity expressed as RU per milliliter of final volume. Each activation mixture was placed in a water bath at 25 C and 1-ml portions removed at regular intervals for activity determinations. Pepsin-catalyzed activation at p H 5.5 and 6.0 was compared with activation of control samples at p H 5.0, 5.5, and 6.0. Results of this experiment are shown in Fig. 4. In the absence of pepsin, activation at p H 6.0 was practically nonexistent and, although pepsin increased the rate of activation at that pH, it still was not complete after 95 hr. A t p H 5.5, activation proceeded more rapidly, particularly when catalyzed by pepsin. A n increase in the level of pepsin decreased the activation time. Addition of pepsin equivalent to six units of milk-clotting activity per milliliter of activation mixture at p H 5.5 produced maximum activity in 45 hr, about the time required for activation at p H 5.0 without added pepsin. Kinetics of pepsin-catalyzed activation. Activation curves for two samples of prorennin activated at p H 6.0 by two and four milk5 Supplied by Dairyland Food Laboratories, Waukesha, Wisconsin.
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FIQ. 4. :Effect o~ added pepsin on activation of crude rennet extract at 25 C. Ordinary activation (O pH 5.0; f-1 pH 5.5; ,,pH 6.0). Pepsin-catalyzed activation ( • pH 5.5, 2 RU pepsin; • p H 5.5, 4 RU pepsin; X pH 5.5, 6 RU pepsin; • pH 6.0, 4 RU pepsin; A pH 6.0, 6 RU pepsin). clotting units of pepsin per milliliter are shown in Fig. 5. During the initial stages of activation, both samples appeared to follow zero-order kinetics, as indicated by the straight lines. However, as the reaction proceeded, the activation rates of both samples decreased markedly, but the changes did not occur at the same level of activity. Discussion
The activation of prorennin was catalyzed by pepsin at p H 6.0, where autocatalytic activation was of little consequence. A t lower p H values the rate of pepsin-catalyzed activation increased, but so did that of autocatalytic activation. Addition of 1 ~ NaC1 retarded
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ACTIVATION
1759
activation of prorennin by pepsin at p H 6.0 and 5.5, but the reaction was still faster than in rennin-catalyzed activation mixtures. Above p H 6.0, both pepsin and rennin coagulate milk and liberate a glycoma~ropeptide (GMP) from K-casein (9, 12). The GMP is reportedly devoid of or sufficiently low in aromatic amino acids that there is no significant absorption in the ultraviolet region (13). Below p H 5.5 these enzymes exhibit a more general proteolytic action on casein, as evidenced by their abilities to liberate greater amounts of nonprotein nitrogen with higher absorbancies at 280 m~. The general proteolytic activity of pepsin on casein at low p H values is considerably greater than that of rennin, and in this study was even greater at p H 5.5 and 6.0. There appeared to be a relationship between the ability of pepsin and rennin to catalyze the activation of prorennin and the general proteolyric activities of these enzymes as opposed to their more specific milk-clotting activities. This could explain why pepsin was a better activating catalyst for prorennin at these p H values. Activation of prorennin by two levels of pepsin at p H 6.0 appeared to follow zeroorder kinetics during the early stages of the reactions, then change to substrate-limiting first-order reactions. Since both samples initially contained the same amount of substrate, the change in activation rate could not have been due to rate-limiting substrate concentrations in both samples. This would have required that each sample reach approximately the same level of activity before deviating from its initial rate of activation. A partial explanation may lie in the instability of pepsin at p H 6.0 (3, 16). Deterioration of the activating catalyst was suggested when both samples deviated from their initial activation rates very early in the reaction and at about the same time. A t p i t 5.5, where pepsin was more stable, both samples reached essentially the same activity before there was a major change in their rates of activation (see Fig. 3). However, if pepsin deterioration were the complete answer, one would have expected a gradual decrease in activation rate from the beginning of the reaction. Lack of pepsin stability at p H 6.0 probably complicated the kinetics of the activation reaction. Activation of crude rennet extract with pepsin demonstrated that this process could be applied to commercial rennet manufacture. This would enable activation to take place at a p H where rennin has its greatest stability (between p H 5.0 and 6.0) (18). I t might g. D A I R Y SCIENCE, V O L . 5 1 , NO. 1 1
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RAND AND ERNSTROM
decrease if not eliminate activation losses. The stability of rennin following activation by pepsin is suggested by results shown in Fig. 3. The maximum level of activity achieved in this experiment remained constant f o r two weeks at 25 C. Activation by pepsin would have the disadvantage of an extra cost, but this might be offset by additional milk-clotting activity contributed by the pepsin. In such cases, the p H of the finished extract shou|d be below 5.7 to prevent deterioration of the pepsin during storage. Greater general proteolytic activity by pepsin than by rennin on casein substrates at p t t 6.0 and 5.5 is at variance with studies indicating a slower body breakdown and less soluble nitrogen development in cheese where pepsin was used to replace rennin as a coagulant (17, 21). Since pepsin is unstable above p i t 6.0 (13, 16), an explanation may be that a higher percentage of pepsin than rennin is destroyed during cooking in the cheese vat and, therefore, little of it remains active in the cheese during curing. Studies reporting less proteolysis by pepsin than by rennin in milk samples during long periods of incubation at p H 6.0 and above (17) might also be explained by lack of pepsin stability. References (1) Abrams, A., and C. F. Jacobsen. 1951. Activation of chymotrypsinogen by a proteinase from Bacillus subtilis. Compt. rend. trav. Lab. Carlsberg, Serie Chim., 27:447. (2) Berridge, N. J. 1945. The purification and crystallization of rennin. Biochem. J., 39: 179. (3) Colwick, S. P., and N. O. Kaplan. 1955. Methods in Enzymology. Vol. 2. Academic Press, New York. 987 pp. (4) De Baun, R. M., W. M. Connors, and R. A. Sullivan. 1953. The preparation and crystallization of rennin and a study of some of its properties. Arch. Biochem. Biophys., 43 : 324. (5) Ege, R., and E. Lundsteen. 1934. Ober die Aktivierung des Prochymosins. Biochem. Z., 268 : 164. (6) Ernstrom, C. A. 1958. Heterogeneity of crystalline rennin. J. Dairy Sci., 41:1663. (7) Foltmann, B. 1959. Studies on rennin. II. On the crystallization, stability, and proteolytie activity of rennin. Actm Chem. Scand., 13: 1927.
J. DAIRY sCIENCE "~0L. 51, NO. 11
(8) Greenbaum, L. M., A. Hirshkowitz, and I. Schoichet. 1959. Activation of trypsinogen by Cathepsin B. J. Biol. Chem., 234: 2885. (9) Haberman, W., H. Mattenheimer, H. SkyPeck, and H. Sinohara. 1961. ~3ber die Abspaltung eines Glycomacropeptides bei der Einwirkung yon Pepsin auf Casein bei pH 6.8. Chimia, 15 : 339. (10) Herriott, R. M. 1938. Isolation, crystallization, and properties of swine pepsinogen. J. Gen. Physiol., 21: 501. (11) Hiller, A., J. Plazin, and D. D. Van Slyke. 1948. A study of conditions for Kjeldahl determination of nitrogen and proteins. J. Biol. Chem., 176: 1401. (12) Jo]les, P., and C. Alais. 1960. Etude du Glycopeptide Obtentu par Action de la Presure sur la Casein ~ due Lait de ¥ache. Compt. rend. acad. sei., Paris, 251: 2605. (13) Jolles, P., C. Alais, and J. Jolles. 1961. Etude Comparee des Cascino-G]ycopeptides formes par Action de la Presure sur les Caseines de Vache, de Brebis, et de Chevre. I. Etude de la Pattie peptidique. Biochim. et Biophys. Acta, 51: 309. (14) Kuntiz, M. 1938. Formation of trypsin from trypsinogen by an enzyme produced by a mold of the genus Pvnicilli~n. J. Gen. Physiol., 21: 601. (15) Kunitz, M., and J. H. Northrop. 1936. Isolation from beef pancreas of crystalline trypsinogen, trypsin, a trypsin inhibitor and an inhibitor-trypsin compound. J. Gen. Physiol., 19 : 991. (16) Linklater, P. M. 1961. The significance of rennin and pepsin in rennet. Ph. D. thesis, University of Wisconsin. 53 pp. Univ. Microfilms, Ann Arbor, Michigan [Dissertation Abstrs., 22: 533]. (17) Melachouris, N. P., and S. L. Tuckey. 1964. Comparison of the proteolysis produced by rennet extract and the pepsin preparation metroclot during ripening of Cheddar cheese. J. Dairy Sci., 47: 1. (18) Mickelsen, R., and C. A. Ernstrom. 1967. Factors affecting stability of rennin. J. Dairy Sci., 50: 645. (19) Northrup, J. H., M. Kunitz, and R. M. Herriott. 1948. Crystalline Enzymes. 2nd ed. Columbia Univ. Press, New York. 352 pp. (20) Rand, A. G., and C. A. Ernstrom. 1964. Effect of pH and sodium chloride on activation of prorennin. J. Dairy Sci., 47 : 1181. (21) Sherwood, I. R. 1935. The function of pepsin and rennet in the ripening of Cheddar cheese. J. Dairy Res., 6: 407. (22) Van Slyke, L. L., and J. C. Baker. 1918. The preparation of pure casein. J. Biol. Chem., 35: 127.
Deprtment of Food Science and Industries, University of Wisconsin, Madison Abstract
The ability of pepsin to catalyze the activation of prorennin was demonstrated when purified prorennin solutions buffered at p H 6.0 and 5.5 were activated by pepsin at 25 C with little interference from autocatalytic activation by rennin. Addition of ~ NaC1 retarded activation of prorennin by pepsin at p H 6.0 and 5.5, but the reaction was still faster than in rennincatalyzed activation mixtures containing the same concentration of salt. Pepsincatalyzed activation of prorennin at p H 6.0 appeared to follow zero-order kinetics during the early stages of reaction, followed by a marked change to a reduced rate of activation. However, the kinetics of activation may have been complicated by lack of pepsin stability at p H 6.0. Pepsin was more proteolytic than rennin on casein substrates at all p H values from 2.0 to 6.0. The general proteolytic activities of rennin and pepsin as distinguished from their milk-clotting activities may be related to their respective abilities to activate prorennin. Pepsin was able to stimulate activation of crude rennet extracts at p H 5.5, where rennin has good stability. Use of pepsin in commercial rennet activation could reduce or eliminate activation losses. The common proteolytlc enzymes found in the digestive tract of animals are secreted as inactive precursors. Activation is usually accomplished by a proteolytic splitting of the z3~nogens into active enzymes and peptide fragments. Proreunin is the predominant enzymatic secretion of the abomasum of young calves, and in the acid environment of the stomach is rapReceived for publication June 12, 1967. 1 Published with approval of the Director, Wisconsin Agricultural Experiment Station. This study was supported in part by a research grant from the Research Committee of the Graduate School, University of Wisconsin, from funds supplied by the Wisconsin Alumni Research Foundation. 3Present address: Department of Animal Science, University of Rhode Island, Kingston. "Present address: Department of Food Science and Industries, Utah State University, Logan.
idly converted into rennin. The activation reaction is mainly autocatalytic, in which rennin attacks prorennin to produce more rennin (20). Above p H 6.0, activation is insignificant. On the other hand, rennin has excellent milkclotting activity at p H 6.7. Literature coneerning rennin action on casein suggests that the enzyme may function differently at different p H values. Between p H 3.0 and 4.0, it has maximum general proteolytic activity, and gives rise to noncasein nitrogen compounds which contain aromatic amino acids (2, 4, 7). The milk-clotting activity of rennin above p H 6.0 is attributed to its ability to liberate a glycomacropeptide from casein. Under these conditions, hydrolysis is rather specific and the liberated peptides contain few aromatic amino acids capable of absorbing ultraviolet light (13). This suggests that the ability of rennin to catalyze the activation of prorennin might be associated with its general proteolytic function and not with its specialized milk-clotting activity. I f this is true, other general proteolyric enzymes might also catalyze the activation of prorennin. As a calf grows, and consumes i n c r e a s i n g quantities of solid feed, pepsin gradually replaces rennin as the main proteolytic enzyme of the stomach. Consequently, it is not surprising that pepsin is found in some commercial renent extracts (16) ; and it is a matter of interest to know whether and under what conditions it will catalyze the activation of prorennin. Zymogens such as pepsinogen and trypsinogen normally activated by autocatalysis (10, 15) can also be activated by other proteolytic enzymes (8, 14, 19). Activation of chymotrypsinogen can be catalyzed by trypsin (19) or by a protease from Bacillus subtilis (1). Ege and Lundsteen (5) reported that pancreatin would activate prorennin in rennet extracts above p I I 5.5. Linklater (16) used pepsin to increase the activation rate of purified prorennin at p H 4.7 in solutions containing 15% sodium chloride. However, it is known that considerable autocatalytic activation of prorennin can also occur under these conditions (20). Conclusive evidence of the ability of pepsin to activate prorennin would require conditions under which autoeatalytic activation by rennin could be blocked or markedly inhibited without producing a similar effect on the action of pep-
1756
PRORENNIN ACTIVATION sin. The purpose of this study was to find such conditions, and to investigate the characteristics of the pepsin-catalyzed activation reaction. Experimental Methods
1757
filter paper had been washed in several changes of distilled water to remove residues capable of absorbing light at 280 m~. TCA filtrates were analyzed for nonprotein nitrogen (NPN) and for absorbancy at 280 m~. Blanks were prepared at each p H for both enzymes by adding 5 ml of buffered 3% casein solution to 1 ml of 72% TCA, followed by 0.1 ml of the enzyme solution. A f t e r 10 rain the precipitate was removed by filtration, and blank values for N P N and absorbaney determined on the filtrate. Activation of prorennin. Solutions of pepsin and rennin were tested for milk-clotting activity, and diluted so that 1 ml would give the desired activity in the final activation mixture. Activation mixtures were prepared by adding ] ml of diluted enzyme solutions to 15 ml of 0.3 ~ sodium citrate buffer at p H 5.5 or 6.0, followed by the addition of 2 ml of a concentrated prorennin solution. I n control samples the enzyme was replaced by an equivalent volume of distilled water. Samples were removed at regular intervals and tested for milkclotting activity.
Enzyme activity. Milk-dotting activities of both rennin and pepsin were determined as previously described (6). The definition of a unit of milk-clotting activity rennin unit (RU) was also reported earlier (20). The same definition was applied to the milk-clotting activity of pepsin. Nitrogen analysis. Nitrogen was measured by a semimicro-Kjeldahl procedure (11). Enzymes. Crystalline rennin was prepared by a method already described (6). Three-timescrystallized pepsin was obtained from Pentex Inc., Kankakee, Illinois. Purified prorennin was isolated from ealf-stomach tissue by a salt fractionation and D E A E chromatographic procedure (20). Casein digestion. Acid casein was prepared according to the method of Van Slyke and Baker (22), and dried with acetone. Two 6% stock solutions of casein were prepared, one Results A comparison of the hydrolytic actions of at p H 6.0 and another at p H 2.5. Separate portions of dry casein were mixed with small rennin and pepsin in casein substrates between amounts of distilled water, placed in an ice p i t 2.0 and 7.0 is shown in Fig. 1. Results are bath, and constantly agitated with a magnetic plotted in terms of nonprotein nitrogen and stirrer. Sodium hydroxide (0.1 ~ ) was added absorbaney at 280 in~ in 12% TCA filtrates. Above p H 5.5, rennin liberated nonprotein to one chilled suspension and agitated until the casein was dissolved at p i t 6.0. The second was nitrogen from casein, but very little of this similarly adjusted to p H 2.5 with 0.1 ~ HC1, material absorbed light at 280 m~. TCA filand agitated until solution was achieved. Each trates from samples treated with pepsin at p H solution was diluted to the required volume with 5.5 and 6.0 not only contained more nonprotein distilled water. Aliquots of the stock solutions nitrogen than those treated with rennin, but were then diluted with equal quantities of 0.3 M buffer to give final casein concentrations of Z © 3%. Buffers used were p H 2.0, phosphate; p H - - absorbancy Z ~'~ --nonprotein n;trogen 3.0, citrate; p H 4.0, lactate; p H 5.0, 5.5, and -U "~%~ ~ rennin pepsin 6.0, citrate; p H 6.5 and piE[ 7.0, phosphate. 2.0 0.2 0 -4 The stock casein solution at p i t 2.5 was mixed o rq Cq with buffers from p H 2.0 to 4.0; the stock z solution at p H 6.0 was mixed with the buffers / ~ \\ \ -4 >from p H 5.0 to 7.0. o / \ \ Solutions of crystalline rennin and pepsin 0 10 o.1 orq were prepared so that the addition of 0.1 ml z of enzyme to 5 ml of a buffered 3% casein frsolution would give a final activity equivalent O to 1 RU per milliliter in the reaction mixtures. The enzymes were allowed to act for I hr 0 0 at 25 C. The reaction was stopped by the 2.0 3.0 4.0 5.0 5,0 ZO addition of sufficient 72% trichloroacetie acid pH (TCA) to give a final concentration of 12% FIG. 1. Effect of p i t on nonprotein nitrogen in the reaction mixture. Ten minutes after the and absorbancy (280 m~) of 12% trieh]oroacetic reaction was stopped, the mixture was filtered acid filtrates following digestion of casein with through no. 42 Whatman filter paper. The pepsin and rennin for 1 hr at 25 C. J . D A I R Y S C I E N C E , VOL. 51, NO. 1 1
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A N D ERNST]~01~I
they also had higher absorbancy values. The proteolytic optimum for the action of rennin on casein appeared to lie near p H 4.0, as previously reported by Foltmann (7). The proteolytic activity of pepsin increased as the p H was reduced to 2.0. Even though its optimum p H for the digestion of casein was well below that of rennin, pepsin still exhibited greater general proteolytie activity than rennin at p H 5.5 and 6.0. Activation of prorennin by pepsin. Activation mixtures containing prorennin at p H 6.0 were treated with two and four milk-clotting units of pepsin, respectively. Other samples were treated with zero and two milk-clotting units (RU) of rennin for comparison. Activation curves for each sample are shown in Fig. 2. A rapid initial increase in activity followed by transition to a more gradual rate of activation occurred in the prorennin samples containing added pepsin. More than 90% of the potential reninn was activated in 360 hr, while autocatalytic activation resulted in the activation of only 20% of the potential rennin in about 300 hr. Addition of rennin had little effect on rate of activation. The slightly higher activity was owing to added rennin. Although the experiment at p H 6.0 clearly demonstrated that pepsin could catalyze the activation of prorennin, rate of activation was extremely slow. Therefore, a similar experiment was conducted at p H 5.5. Results are presented in Fig. 3. Activation of samples containing added pepsin proceeded rapidly at first, then continued more slowly until maximum activity was reached after about 50 hr. Autocatalytic activation by rennin, although somewhat faster than at p H 6.0, resulted in only 45% of maximum activity in 180 hr and did not bring about complete activation even after 360 hr. Rennin had a greater catalytic effect at p H 5.5 than at p H 6.0, as demonstrated by the slightly higher rate of autocatalytic activation.
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Fie. 3. Pepsin- and rennin-catalyzed activation of prorennin at pH 5.5 in 0.3 ~ sodium citrate buffer at 25 C. Addition of 1~ NaC1 to activation mixtures retarded pepsin-catalyzed activation of prorennin at both p H 6.0 and 5.5. However, it was still considerably faster than in rennin-catalyzed controls containing the same concentration of salt.
Activation of crude rennet extracts by pepsin. Unactivated commercial extract s at 2 C was divided into several 300-ml portions and each one adjusted to the desired p H with 1 N lactic acid or disodium phosphate. One milliliter of pepsin solution was then added to 20 ml of the cold extract. Distilled water was substituted for pepsin in control samples. Pepsin solutions were prepared to provide the desired milkclotting activity expressed as RU per milliliter of final volume. Each activation mixture was placed in a water bath at 25 C and 1-ml portions removed at regular intervals for activity determinations. Pepsin-catalyzed activation at p H 5.5 and 6.0 was compared with activation of control samples at p H 5.0, 5.5, and 6.0. Results of this experiment are shown in Fig. 4. In the absence of pepsin, activation at p H 6.0 was practically nonexistent and, although pepsin increased the rate of activation at that pH, it still was not complete after 95 hr. A t p H 5.5, activation proceeded more rapidly, particularly when catalyzed by pepsin. A n increase in the level of pepsin decreased the activation time. Addition of pepsin equivalent to six units of milk-clotting activity per milliliter of activation mixture at p H 5.5 produced maximum activity in 45 hr, about the time required for activation at p H 5.0 without added pepsin. Kinetics of pepsin-catalyzed activation. Activation curves for two samples of prorennin activated at p H 6.0 by two and four milk5 Supplied by Dairyland Food Laboratories, Waukesha, Wisconsin.
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FIQ. 4. :Effect o~ added pepsin on activation of crude rennet extract at 25 C. Ordinary activation (O pH 5.0; f-1 pH 5.5; ,,pH 6.0). Pepsin-catalyzed activation ( • pH 5.5, 2 RU pepsin; • p H 5.5, 4 RU pepsin; X pH 5.5, 6 RU pepsin; • pH 6.0, 4 RU pepsin; A pH 6.0, 6 RU pepsin). clotting units of pepsin per milliliter are shown in Fig. 5. During the initial stages of activation, both samples appeared to follow zero-order kinetics, as indicated by the straight lines. However, as the reaction proceeded, the activation rates of both samples decreased markedly, but the changes did not occur at the same level of activity. Discussion
The activation of prorennin was catalyzed by pepsin at p H 6.0, where autocatalytic activation was of little consequence. A t lower p H values the rate of pepsin-catalyzed activation increased, but so did that of autocatalytic activation. Addition of 1 ~ NaC1 retarded
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ACTIVATION
1759
activation of prorennin by pepsin at p H 6.0 and 5.5, but the reaction was still faster than in rennin-catalyzed activation mixtures. Above p H 6.0, both pepsin and rennin coagulate milk and liberate a glycoma~ropeptide (GMP) from K-casein (9, 12). The GMP is reportedly devoid of or sufficiently low in aromatic amino acids that there is no significant absorption in the ultraviolet region (13). Below p H 5.5 these enzymes exhibit a more general proteolytic action on casein, as evidenced by their abilities to liberate greater amounts of nonprotein nitrogen with higher absorbancies at 280 m~. The general proteolytic activity of pepsin on casein at low p H values is considerably greater than that of rennin, and in this study was even greater at p H 5.5 and 6.0. There appeared to be a relationship between the ability of pepsin and rennin to catalyze the activation of prorennin and the general proteolyric activities of these enzymes as opposed to their more specific milk-clotting activities. This could explain why pepsin was a better activating catalyst for prorennin at these p H values. Activation of prorennin by two levels of pepsin at p H 6.0 appeared to follow zeroorder kinetics during the early stages of the reactions, then change to substrate-limiting first-order reactions. Since both samples initially contained the same amount of substrate, the change in activation rate could not have been due to rate-limiting substrate concentrations in both samples. This would have required that each sample reach approximately the same level of activity before deviating from its initial rate of activation. A partial explanation may lie in the instability of pepsin at p H 6.0 (3, 16). Deterioration of the activating catalyst was suggested when both samples deviated from their initial activation rates very early in the reaction and at about the same time. A t p i t 5.5, where pepsin was more stable, both samples reached essentially the same activity before there was a major change in their rates of activation (see Fig. 3). However, if pepsin deterioration were the complete answer, one would have expected a gradual decrease in activation rate from the beginning of the reaction. Lack of pepsin stability at p H 6.0 probably complicated the kinetics of the activation reaction. Activation of crude rennet extract with pepsin demonstrated that this process could be applied to commercial rennet manufacture. This would enable activation to take place at a p H where rennin has its greatest stability (between p H 5.0 and 6.0) (18). I t might g. D A I R Y SCIENCE, V O L . 5 1 , NO. 1 1
1760
RAND AND ERNSTROM
decrease if not eliminate activation losses. The stability of rennin following activation by pepsin is suggested by results shown in Fig. 3. The maximum level of activity achieved in this experiment remained constant f o r two weeks at 25 C. Activation by pepsin would have the disadvantage of an extra cost, but this might be offset by additional milk-clotting activity contributed by the pepsin. In such cases, the p H of the finished extract shou|d be below 5.7 to prevent deterioration of the pepsin during storage. Greater general proteolytic activity by pepsin than by rennin on casein substrates at p t t 6.0 and 5.5 is at variance with studies indicating a slower body breakdown and less soluble nitrogen development in cheese where pepsin was used to replace rennin as a coagulant (17, 21). Since pepsin is unstable above p i t 6.0 (13, 16), an explanation may be that a higher percentage of pepsin than rennin is destroyed during cooking in the cheese vat and, therefore, little of it remains active in the cheese during curing. Studies reporting less proteolysis by pepsin than by rennin in milk samples during long periods of incubation at p H 6.0 and above (17) might also be explained by lack of pepsin stability. References (1) Abrams, A., and C. F. Jacobsen. 1951. Activation of chymotrypsinogen by a proteinase from Bacillus subtilis. Compt. rend. trav. Lab. Carlsberg, Serie Chim., 27:447. (2) Berridge, N. J. 1945. The purification and crystallization of rennin. Biochem. J., 39: 179. (3) Colwick, S. P., and N. O. Kaplan. 1955. Methods in Enzymology. Vol. 2. Academic Press, New York. 987 pp. (4) De Baun, R. M., W. M. Connors, and R. A. Sullivan. 1953. The preparation and crystallization of rennin and a study of some of its properties. Arch. Biochem. Biophys., 43 : 324. (5) Ege, R., and E. Lundsteen. 1934. Ober die Aktivierung des Prochymosins. Biochem. Z., 268 : 164. (6) Ernstrom, C. A. 1958. Heterogeneity of crystalline rennin. J. Dairy Sci., 41:1663. (7) Foltmann, B. 1959. Studies on rennin. II. On the crystallization, stability, and proteolytie activity of rennin. Actm Chem. Scand., 13: 1927.
J. DAIRY sCIENCE "~0L. 51, NO. 11
(8) Greenbaum, L. M., A. Hirshkowitz, and I. Schoichet. 1959. Activation of trypsinogen by Cathepsin B. J. Biol. Chem., 234: 2885. (9) Haberman, W., H. Mattenheimer, H. SkyPeck, and H. Sinohara. 1961. ~3ber die Abspaltung eines Glycomacropeptides bei der Einwirkung yon Pepsin auf Casein bei pH 6.8. Chimia, 15 : 339. (10) Herriott, R. M. 1938. Isolation, crystallization, and properties of swine pepsinogen. J. Gen. Physiol., 21: 501. (11) Hiller, A., J. Plazin, and D. D. Van Slyke. 1948. A study of conditions for Kjeldahl determination of nitrogen and proteins. J. Biol. Chem., 176: 1401. (12) Jo]les, P., and C. Alais. 1960. Etude du Glycopeptide Obtentu par Action de la Presure sur la Casein ~ due Lait de ¥ache. Compt. rend. acad. sei., Paris, 251: 2605. (13) Jolles, P., C. Alais, and J. Jolles. 1961. Etude Comparee des Cascino-G]ycopeptides formes par Action de la Presure sur les Caseines de Vache, de Brebis, et de Chevre. I. Etude de la Pattie peptidique. Biochim. et Biophys. Acta, 51: 309. (14) Kuntiz, M. 1938. Formation of trypsin from trypsinogen by an enzyme produced by a mold of the genus Pvnicilli~n. J. Gen. Physiol., 21: 601. (15) Kunitz, M., and J. H. Northrop. 1936. Isolation from beef pancreas of crystalline trypsinogen, trypsin, a trypsin inhibitor and an inhibitor-trypsin compound. J. Gen. Physiol., 19 : 991. (16) Linklater, P. M. 1961. The significance of rennin and pepsin in rennet. Ph. D. thesis, University of Wisconsin. 53 pp. Univ. Microfilms, Ann Arbor, Michigan [Dissertation Abstrs., 22: 533]. (17) Melachouris, N. P., and S. L. Tuckey. 1964. Comparison of the proteolysis produced by rennet extract and the pepsin preparation metroclot during ripening of Cheddar cheese. J. Dairy Sci., 47: 1. (18) Mickelsen, R., and C. A. Ernstrom. 1967. Factors affecting stability of rennin. J. Dairy Sci., 50: 645. (19) Northrup, J. H., M. Kunitz, and R. M. Herriott. 1948. Crystalline Enzymes. 2nd ed. Columbia Univ. Press, New York. 352 pp. (20) Rand, A. G., and C. A. Ernstrom. 1964. Effect of pH and sodium chloride on activation of prorennin. J. Dairy Sci., 47 : 1181. (21) Sherwood, I. R. 1935. The function of pepsin and rennet in the ripening of Cheddar cheese. J. Dairy Res., 6: 407. (22) Van Slyke, L. L., and J. C. Baker. 1918. The preparation of pure casein. J. Biol. Chem., 35: 127.