Assay of the Esterase Activity of Thrombin, Plasmin and Trypsin with a Chromogenic Substrate p-Nitrobenzyl p-Toluenesulfonyl-L-Arginine

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Abstract
P-Nitrobenzyl p-toluenesulfonyl-L-arginine has been synthesized. A number of trypsin-like enzymes can catalyze the hydrolysis of this ester leading to formation of p-nitrobenzyl alcohol. After separation from the ester and p-toluenesulfonylarginine by extraction into chloroform, the p-nitrobenzyl alcohol liberated can be measured spectrophotometrically at 271 nm. Under the conditions of the assay, the hydrolysis of 1 μmol/ml of the ester is equivalent to an absorbance change of 4.45 cm–1 at 271 nm. With 0.10 mM p-nitrobenzyl p-toluenesulfonyl-L-arginine in 0.1 M Tris-HCl at pH 8.4 and 30°, the enzymatic hydrolysis is linearly proportional to time up to consumption of 60% of the ester. Product formation is proportional to enzyme concentration with 0.05 to 0.2 NIH clotting units/ml for bovine or human thrombin, 0.005 to 0.02 CTA units/ml for human plasmin, and 0.01 to 0.04 μg/ml protein for bovine pancreatic trypsin. In 0.1 M Tris-HCl at pH 8.4 and 30°, Kmis 14 μM and Vmax is 0.037 μmol/min/NIH unit/ml for bovine thrombin, Km is 78 μM and Vmax is 0.31 μmol/min/CTA unit/ml for human plasmin, and Km is 12 μM and Vmax is 138 μmol/min/mg protein/ml for bovine trypsin. With bovine thrombin, activities at pH 7.3 and at pH 9.2 were 30% lower and 40-50% higher than the rate at pH 8.4. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2133 NIH units/mg protein showed esterase activities varying from 0.15 to 0.4 μmol p-nitrobenzyl alcohol formed/10 min/NIH unit.