The inhibition of trypsin. IV. Reaction with diethyl p-nitrophenyl phosphate in the presence of urea.

The inhibition of proteolytic enzymes by organophosphorus compounds has been extensively studied in recent years (1, 2). Such studies have shown that the reaction of trypsin with diisopropyl fluorophosphate (3) or diethyl p-nitrophenyl phosphate (DENP) (4) is accompanied by a loss in the proteolytic, esterase, and amidase activities of this enzyme. It is not certain, however, whether these inhibitors combine at the site responsible for the catalytic function of trypsin (4, 5). In a previous paper in this series (6), it was shown that, although trypsin was inactivated by concentrations of urea ranging from 3 to 5 M, its enzymatic activity was retained in 6 to 8 M urea solution, Since most proteins are known to undergo rapid denaturation in high concentrations of urea, a more detailed examination has been made of the properties of trypsin in 8 M urea. During the course of such studies it was noted that, unlike native trypsin, the proteolytic activity of urea-treated trypsin was not inhibited by DENP. This observation prompted the present investigation, the object of which was to determine whether DENP reacts with trypsin at a site which differs from the site responsible for the catalytic function of the enzyme.