TEMPORARY INHIBITION OF TRYPSIN*

TEMPORARY INHIBITION OF TRYPSIN* BY M. LASKOWSKI AND FENG CHI WU (From the Department oj Biochemistry, Marquette University School of Medicine, Milwaukee, Wisconsin) (Received for publication, April 30, 1953) In the previous work dealing with naturally occurring trypsin inhibitors it has been generally assumed or at least implied that as long as the inhibitor is capable of reacting with trypsin the inhibitor is resistant to tryptic digestion. In the present paper a system is described in which trypsin is first inactivated by the inhibitor and then released, owing to the digestion of the inhibitor. The term temporary inhibition is suggested for this phenomenon. EXPERIMENTAL Trypsin was prepared by the method of Kunitz and Northrop (1) as modified by McDonald and Kunitz (2), and was recrystallized three times. The inhibitor was prepared by the method of Kazal, Spicer, and Brahinsky (3) and was a gift from Dr. L. A. Kazal, to whom we express our deep gratitude. Tryptic activity and the inhibitory activity were determined by the method of Kunitz (4). Since the preparation of trypsin was slowly losing activity during the period of this work, the values of trypsin are expressed both in actual micrograms of the protein and in units of tryptic activity, as found on the day of the experiment. Other inhibitors were also tried: pancreatic trypsin inhibitor prepared according to Kunitz and Northrop (1) ; soy bean inhibitor (5) purchased from the Worthington Biochemical Laboratory; colostrum inhibitor prepared by the method of Laskowski et al. (6, 7) ; and partially purified blood plasma inhibitor, prepared by the method of Peanasky and Laskowski (8). The starting point for the present investigation was the experiment summarized in Table I. This was devised as a continuation of the work of Mars et al. (9), who attempted to check the hypothesis of Haanes and Gyijrgy (10) that enterokinase is capable of overcoming the action of the trypsin inhibitor on trypsin. Mars et al. (9) used the crystalline trypsin-trypsin inhibitor complex of Kunitz and Northrop (1) and were unable to confirm the hypothesis of Haanes and Gyorgy (10). It was felt, however, that Mars et al. did not exclude the possible action of enterokinase on the com- * Aided by a research grant from the National Institutes of Health, United States Public Health Service. 797

798 INHIBITION OF TRYPSIN plex of trypsin with Kazal s inhibitor. Such a complex conceivably could have been present in the lipotropic fraction used by Haanes and Gyijrgy (10) and prepared by Bosshardt et al. (11) from the residue which remained after the extraction of pancreas for insulin. The results shown in Table I indicate that, in agreement with previous findings (9) on the pancreatic complex of Kunitz and Northrop, enterokinase did not influence the dissociation of the complex composed of trypsin and Kazal s inhibitor. Appearance of free trypsin, however, was noticed in both tubes containing calcium, regardless of their content of enterokinase. TABLE Effect of Calcium on Apparent Dissociation of Trypsin-Kazal s Trypsin Inhibitor Complex Time 15 sec. 30 min. 60 3 hrs. 7 24 Tube 1. Calcium Tube 3. enterokinase Tube 2. Calcium Enterokinase I Tube 4 0.093 0.060 0.075 0.048 0.039 0.030 0.045 0.031 0.040 0.043 0.035 0.027 0.070 0.070 0.043 0.037 0.120 0.115 0.051 0.032 0.277 0.267 0.076 0.054 All tubes contained 4.0 ml. of 0.1 M phosphate buffer at ph 7, 1.0 ml. of trypsin solution containing 100 y per ml., 1.0 ml. of Kazal s inhibitor solution containing 40 7 per ml., and water to bring the volume to 10 ml. In addition, Tubes 1 and 2 contained 0.1 ml. of 1 M CaClz, and Tubes 1 and 3 contained 160 y of enterokinase. All tubes were mixed and allowed to stand at room temperature. At the indicated time, 1 ml. aliquots were withdrawn and analyzed for trypsin according to Kunits (4). All figures are expressed as optical density at 280 mp and are corrected for the values of blanks. Since the protective action of calcium on trypsin had been shown by Gorini (12) and by Nord and coworkers (13, 14), it was postulated that the liberation of trypsin occurred in all tubes, but was evidenced only in the tubes containing calcium, which protected trypsin from denaturation and self-digestion. This hypothesis was verified by the experiment illustrated in Fig. 1. The initial amounts of trypsin and inhibitor (Curves I and II) were so chosen as to leave, after mixing, about 20 per cent of the trypsin free. The results indicated that the liberation of trypsin occurred in both tubes, with and without calcium. The amount of apparently liberated trypsin was, however, much higher in the presence of calcium. The rates of inactivation of trypsin in the presence and in the absence of calcium are also

M. LASKOWSKI AND F. C. WU 799 indicated on Fig. 1. The inhibitor alone was stable under these conditions, with or without calcium. The results of an experiment in which varying concentrations of calcium were used are illustrated in Fig. 2. As in the previous experiment, the amounts of inhibitor were insufficient to inhibit all of the trypsin present. The results indicate that the height of the peaks, representing tryptic activity, increased with increasing concentration of calcium. On the other FIG. 1 FIG. 2 FIG. 1. The effect of calcium on the apparent liberation of trypsin from the complex. All tubes contained 10 y per ml. of trypsin (equivalent to 27.0 tryptic units of Kunitz per ml.) and borate buffer at ph 7.0, final concentration 0.04 M. In addition Tube 1 (Curve I) contained Kazal s inhibitor 2.4 y per ml. and CaClz, final concentration 0.01 M; Tube 2 (Curve II) contained Kazal s inhibitor 2.4 y per ml. but no calcium; Tube 3 (Curve III) contained 0.01 M CaClz but no inhibitor; Tube 4 (Curve IV) contained neither calcium nor inhibitor. Exposed at room temperature of about 25. FIG. 2. The effect of varying concentrations of calcium on the apparent liberation of trypsin from the complex. All tubes contained 10 y per ml. of trypsin (29.6 units per ml.), 2.0 y per ml. of Kazal s inhibitor, 0.04 M borate buffer at ph 7.0, and the indicated amounts of CaClz: 0, 0.02 M; 0, 0.01 M; 0, 0.005 M; 0, 0.001 M. Exposed at room temperature. hand, the maximal value for each tube was reached sooner in the tubes containing lower concentrations of calcium. Fig. 3 illustrates the results of an experiment in which the amounts of trypsin and calcium were kept constant, but the amount of inhibitor was varied. The findings indicate that the maximal value of the liberated trypsin was reached faster in the tubes containing lower amounts of the inhibitor. The highest peak was reached in the tube containing the largest amount of the inhibitor. It was then postulated that the appearance of the free trypsin was due to the digestion of the inhibitor. This hypothesis was verified by the ex-

800 INHIBITION OF TRYPSIN periment illustrated in Fig. 4. The inhibitor of Kazal et al. (free or in the form of a complex) can be heated to 80 for 5 minutes in a solution containing 2.5 per cent trichloroacetic acid without denaturation, while trypsin is denatured by this procedure. After cooling and neutralization, the inhibitor remaining in the solution can be determined by mixing it with a fresh solution of trypsin. In the experiment illustrated in Fig. 4, trypsin and inhibitor were mixed and left at room temperature. As in the previous experiment, the amount FIG. 3 FIG. 4 FIG. 3. The effect of varying concentrations of inhibitor on the liberation of trypsin. All tubes contained 10 y per ml. of trypsin (27.6 units per ml.), 0.01 M CaC12, 0.04 M borate buffer at ph 7.0, and the indicated amounts of Kazal s inhibitor: 0, 1.2~perml.;0,1.6~perml.; l,2.0rperml.; 0,2.4rperml. FIG. 4. Comparison of the appearance of trypsin with disappearance of inhibitor. All tubes contained 0.01 M CaCla and 0.04 M borate buffer at ph 7.0. The experimental tube (0, 0) contained 10 y per ml. of trypsin (22.0 units per ml.) and 1.6 y per ml. of Kazal s inhibitor; 0, tryptic activity, 0, inhibitory activity expressed as trypsin inhibited. Control tube 0 contained 10 y per ml. of trypsin only; control tube 0 contained 1.6 y per ml. of Kazal s inhibitor only. Exposed at room temperature. of inhibitor was not sufficient to inactivate all of the trypsin. At the indicated intervals, aliquots were withdrawn, and both the appearance of trypsin and disappearance of inhibitor were determined on separate aliquots. It is evident that the amount of the free trypsin appearing corresponded to the amount of inhibitor disappearing. The degree of self-digestion of trypsin and the stability of the solution of pure inhibitor, under the conditions of the experiment, are also indicated in Fig. 4. When the amount of trypsin which appeared was corrected for self-digestion of trypsin, the line illustrated in Fig. 5 was obtained. The points were plotted to indicate the amount of trypsin which should have appeared, calculated from the amounts of digested inhihit,or found. The agreement was good.

M. LASKOWSKI AND F. C. WU 801 A few additional points required clarification. Under optimal conditions for the determination of inhibitory activity, Laskowski el al. (7) found that Kazal s inhibitor reacted stoichiometrically with trypsin. The determinations, however, were never carried to the region where all the trypsin was inactivated. An experiment was therefore performed in which the inhibitor, casein, and the required amounts of water were placed in the analytical tubes and trypsin was pipetted directly into these tubes at zero time. By doing so, the effect of digestion was minimized, but was not entirely FIG. 5 INHIBITOR SOLU T I ON FIG. 6 FIG. 5. Comparison of the amounts of the trypsin appearing, calculated from values (0) of trypsin observed after correcting for self-digestion of kypsin and (0) calculated from the amounts of digested inhibitor. The calculations were made from the data shown in Fig. 4. FIG. 6. The effect of varying concentrations of trypsin and of inhibitor on the degree of inhibition. The solution of Kazal s inhibitor contained 4 y per ml., 15 y of trypsin (0) (40.4 units), 10 -y of trypsin (0) (25.2 units), 5 y of trypsin ((3) (13.4 units) per tube. All other conditions as in the assay of Kunitz (4). eliminated, since the standard incubation of 20 minutes required by the procedure of Kunitz could not be avoided. The results (Fig. 6) indicate that the amount of dissociation was in no case higher than 5 per cent. The formation of a complex between trypsin and Kazal s inhibitor was found to be a very rapid process. Whether the observed value of about 5 per cent should be considered as a true value of dissociation cannot be definitely stated. No attempts were made to find a method fast enough either to ascertain that the equilibrium had been reached or to exclude the possibility of digestion. In view of the above finding, an experiment, analogous to that shown in Fig. 4, was performed. The amounts of trypsin and inhibitor were so

802 INHIBITION OF TRYPSIN chosen as to inhibit all of the trypsin and leave a small excess of the free inhibitor. Both the appearance of trypsin and the disappearance of inhibitor were followed on different aliquots withdrawn at the indicated time. The results are illustrated in Fig. 7. The zero time samples represent the aliquots withdrawn at about 1 minute after mixing. The tubes were exposed at 37 and were covered with a layer of toluene. It was previously established that covering with toluene did not exert any detectable influ- 0 20 40 60 80 100 FIG. 7 FIG. 8 FIG. 7. Comparison of the appearance of trypsin with the disappearance of inhibitor. All tubes contained 0.01 M CaClz and 0.04 M borate buffer at ph 7.0. The experimental tube (0, 0) contained 10 y per ml. of trypsin (25.7 units per ml.) and 2.4 y per ml. of Kazal s inhibitor; 0, tryptic activity; 0, inhibitory activity expressed as trypsin inhibited. The control tube (a) contained 10 y per ml. of trypsin only. Exposed at 37. FIG. 8. The effect of varying concentrations of the inhibitor on the rate of disappearance of the inhibitor, expressed in tryptic units. All tubes contained 0.01 M CaCL, 0.04 M borate buffer at ph 7.0, 10 y per ml. of trypsin (21.5 units per ml.), and the indicated amounts of Kazal s inhibitor: 0,4.8 y per ml., l,3.2 y per ml., 0,2.4 y per ml., 0, 0.8 y per ml. Exposed at 37. HF. ence on either of the components of the system. As would be expected from the results shown in Fig. 6, even in the first sample a small amount of tryptic activity was detected. The digestion of the inhibitor was accomplished within 22 hours. During the period of digestion of the inhibitor (Fig. 7), tryptic activity increased and then started decreasing slowly. After 95 hours, the amount of tryptic activity that was left in the tube originally containing inhibitor was more than 1.5 times that in the tube containing trypsin alone. An experiment was then performed in which the quantity of trypsin was kept constant, but the amount of inhibitor was varied from 50 to 300 per

M. LASKOWSKI AND F. C. WU 803 cent of the amount necessary to inhibit all of the trypsin (Fig. 8). Only the disappearance of the inhibitor was measured. In spite of the longer and more involved analytical procedure, this was chosen in preference to measurement of the appearance of trypsin, since it was felt that correcting for the self-digestion of trypsin would not be accurate enough, in view of changing concentrations. The results (Fig. 8) indicate that the velocity of the digestion of the inhibitor was influenced by the initial concentration of the inhibitor. The rate of digestion of inhibitor was slower in the tubes in which the ratio of inhibitor to trypsin was higher. Finally, we wished to determine whether the inhibitor of Kazal et al. is unique in its properties of being susceptible to tryptic digestion. Several other inhibitors were tested. The pancreatic inhibitor (1) and the inhibitor from colostrum (6), as well as purified inhibitor from blood plasma (8), were found to be resistant during 2 weeks exposure at 37. A small but significant amount of liberation of trypsin was observed with soy bean in- hibitor after 2 weeks exposure at 37 ; the reaction, however, was so slow that no other conclusions were possible. DISCUSSION At present no attempt is made to explain the detailed mechanism of the temporary inhibition of trypsin by Kazal s inhibitor. Two general alternatives have been considered. The simplest is to assume that the inhibitor acts as substrate in the Michaelis-Menten reaction, T + I & TI k3+ T + products kz the value of kl being exceptionally high and that of ks exceptionally low. The alternative would be to assume that two reactions occur, the first of which leads to an inactive complex, T+I&$TI and a second to the observed reaction products, ~1 + T CL TIT A--+ 2T + products Jd where TIT is the Michaelis-Menten complex. In the latter case, it is necessary to assume further that the inactive complex TI and the Michaelis-Menten complex TIT both involve the active centers of trypsin, but different sites on the molecule of the inhibitor. The consequence of the first alternative is that in the rate-limiting re-

804 INHIBITION OF TRYPSIN action the velocity of the formation of products should be a function of [TI] only, dx - dt = k,([ti] - z) while, if the second alternative were true, the velocity of the rate-limiting reaction should be a function of both [TI] and [T] The experimental evidence (Figs. 3 and 8) favors the second alternative. The initial slopes of the curves representing the appearance of trypsin (Fig. 3) are larger in the tubes containing higher initial amounts of trypsin. Even more clearly, the same phenomenon could be seen from the results shown in Fig. 8. The slopes of the lines representing the disappearance of the inhibitor are smaller in the tubes containing a large excess of the inhibitor and increase with increasing ratios of trypsin to inhibitor. One is tempted to speculate on the physiological significance of temporary inhibition. It obviously provides a mechanism by which the already activated trypsin is safeguarded against self-digestion (Figs. 3, 4, and 7). Temporary inhibition slows down and prolongs the action of trypsin, which may be of a particular importance in regulating the speed of activation of chymotrypsinogens. Since thus far only Kazal s inhibitor was found to be susceptible to a reasonably rapid digestion, it seems premature to postulate a wide distribution of similar systems. It does not seem premature, however, to call the attention to a possibility of the existence of similar intracellular systems, which may participate in the regulation of the action of cathepsins. Since tryptic inhibitors including Kazal s inhibitor exert an action against thrombin, it is also possible that similar mechanisms may be involved in blood coagulation. Kazal s inhibitor seems to occupy a unique position among the known trypsin inhibitors, if one disregards the small amounts of digestion noticed with the soy bean trypsin inhibitor. It is interesting to add here that also in respect to action on chymotrypsin 01 Kazal s inhibitor appears to be the only one which does not exert any inhibitory action. SUMMARY A system composed of crystalline trypsin and the crystalline inhibitor of Kazal et al. (3) was described in which trypsin was first inhibited and then slowly released, owing to the digestion of the inhibitor. The term temporary inhibition was suggested for this phenomenon. The probable mechanism of temporary inhibition and its possible physiological significance were discussed. 1 Unpublished.

M. LASKOWSKI AND F. C. WU 805 Addendum-After this manuscript had been submitted for publication, we became aware of the work of Gorini and Audrain (15,16). A year before we submitted our manuscript, these authors (15) found that in a system containing borate buffer, ph 7.9,O.Ol M CaC12, trypsin, and ovomucoid the ovomucoid was rapidly hydrolyzed. While the conclusions of Gorini and Audrain (16) are not identical with ours, the phenomenon they observed strikingly resembles temporary inhibition, indicating that Kazal s inhibitor is not a unique case. BIBLIOGRAPHY 1. Kunitz, M., and Northrop, J. H., J. Gen. Physiol., 19, 991 (1936). 2. McDonald, M. R., and Kunitz, M., J. Gen. Physiol., 29, 155 (1946). 3. Kazal, L. A., Spicer, D. S., and Brahinsky, R. A., J. Am. Chem. Sot., 70, 3034 (1948). 4. Kunitz, M., J. Gen. Physiol., 30, 291 (1947). 5. Kunitz, M., 1. Gen. Physiol., 29, 149 (1946). 6. Laskowski, M., Jr., and Laskowski, M., J. Biol. Chem., 190, 563 (1951). 7. Laskowski, M., Jr., Mars, P. H., and Laskowski, M., J. Biol. Chem., 198, 745 (1952). 8. Peanasky, R. J., and Laskowski, M., J. Biol. Chem., 204, 153 (1953). 9. Mars, P. H., Peanasky, R. J., and Laskowski, M., Proc. Sot. Exp. Biol. and Med., 82, 384 (1953). 10. Haanes, M. L., and Gyorgy, I., Am. J. Physiol., 166, 441 (1951). 11. Bosshardt, D. K., Cieresko, L. S., and Barnes, R. H., Am. J. Physiol., 166, 433 (1951). 12. Gorini, L., Biochim. et biophys. acfa, 7, 318 (1951). 13. Bier, M., and Nord, F. F., Arch. Biochem. and Biophys., 33, 320 (1951). 14. Duke, J. A., Bier, M., and Nord, F. F., Arch. Biochem. and Biophys., 40, 424 (1952). 15. Gorini, L., and Audrain, L., Biochim. et biophys. acta, 8, 702 (1952). 16. Gorini, I,., and Audrain, L., Biochim. et biophys. acta, 10, 570 (1953).