p-Nitrobenzyl p-toluenesulfonyl-L-arginine: a chromogenic substrate for thrombin, plasmin and trypsin

p-Nitrobenzyl p-toluenesulfonyl-L-arginine is hydrolyzed by thrombin, plasmin, and trypsin to p-nitrobenzyl alcohol and tosyl-L-arginine. The absorption of p-nitrobenzyl alcohol formed is measured at 271 nm (AmM 8.89). With 0.10 mM of the ester in 0.1 M Tris-HCl at pH 8.4 and 30 degrees C, the hydrolysis catalyzed by thrombin, plasmin, and trypsin is linearly proportional to time up to consumption of 60% of the substrate. Km is 14 micron and Vmax is 0.037 mumol/min/NIH unit for bovine thrombin, Km is 78 micron and Vmax is 0.31 mumol/min/CTA unit/ml for human plasmin, and Km is 12 micron and Vmax is 138 mumol/min/mg protein/ml for bovine trypsin. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2,133 NIH units/mg protein showed esterase activities ranging from 0.15 to 0.4 mumol p-nitrobenzyl alcohol formed/10 min/NIH unit. Useful ranges for assay of enzymes were (per milliliter): 0.05-0.2 NIH units (thrombin), 0.005-0.02 CTA units (plasmin), and 0.01-0.04 microgram (trypsin).