The behaviour of trypsin towards α-N-methyl-α-N-toluene-p-sulphonyl-l-lysine β-naphthyl ester. A new method for determining the absolute molarity of solutions of trypsin

Abstract

1. α-N-Methyl-α-N-toluene-p-sulphonyl-l-lysine β-naphthyl ester (MTLNE) was synthesized as its hydrobromide and shown to be slowly hydrolysed by bovine pancreatic trypsin. The acylation step, however, is so much faster than deacylation of the acyl-enzyme that spectrophotometric measurement of the ‘burst’ of β-naphthol provides a convenient method for determining the absolute molarity of trypsin solutions. 2. By using the same stock solution of trypsin, application of this method at pH4·0 and pH7·0 as well as that of Bender et al. (1966) at pH3·7 gave concordant results. 3. Provided that [S]₀>[E]₀, the size of the ‘burst’ is independent of substrate concentration. 4. In the trypsin-catalysed hydrolysis of α-N-toluene-p-sulphonyl-l-arginine methyl ester, MTLNE functions as a powerful non-competitive inhibitor. 5. There is no detectable reaction between MTLNE and either bovine pancreatic α-chymotrypsin at pH4·0 or bovine thrombin at pH6·0.