Competition among trypsin inhibitors.

The experiments presented in this paper were designed to elucidate the kinetics of action of three protein inhibitors (from pancreas, soy bean, and egg white) on trypsin, and to discover how the inhibition by these substances was related to the action of diisopropyl fluorophosphate (DFP), mercuric chloride, and calcium salts on trypsin. The most, detailed work on the protein inhibitors of trypsin has been carried out, by Fraenkel-Conrat et al. (1, 2), who investigated the effect of chemical modification of the inhibitor and of trypsin on the combination between the two. They found, for instance, that, although amino groups of trypsin could be largely acetylated (70 per cent) without destruction of the enzyme activity, such acetylation greatly reduced the inhibition of trypsin by ovomucoid (the inhibitor from egg white). This was in agreement, with their limited kinetic data (l), which showed that the inhibition was non-competitive. In addition, Borchers, Ackerson, and Sandstedt (3) had suggested that the inhibition by the soy bean inhibitor was non-competitive, and Green and Work (4) had found the same for the pancreatic inhibitor of Kunitz and Northrop. However, it was pointed out (4) that the Michaelis constant (about lo+ M) of the protein substrates used in all these investigations was so much larger than the dissociation constants of the inhibitor-trypsin compounds (about lo-lo M) that competitive inhibition could not, have been demonstrated by the methods used. In the present work benzoyl+arginine ethyl ester (BAEE) has been used as substrate throughout, since its low Michaelis constant (about lop5 M) (5) makes it, particularly suitable for investigating competition effects. It was shown in a preceding paper (6) that 0.01 M Ca++ produced a 25 per cent increase in the activity of trypsin towards synthetic substrates and this was interpreted in terms of an equilibrium between active and inactive trypsin that was shifted by Ca++. It was therefore of interest to investigate the action of Ca++ on trypsin in the presence of the protein inhibitors. The interactions between the protein inhibitors and diisopropyl fluorophosphate have also been studied, since recognition of the irreversible combination of DFP with the active center of trypsin (7) can be used to