Thiobenzyl benzyloxycarbonyl-l-lysinate, substrate for a sensitive colorimetric assay for trypsin-like enzymes

Thiobenzyl benzyloxycarbonyl-l-lysinate (Z-Lys-SBzl), a substrate for trypsin-likeproteases, was synthesized. In the presence of 5,5′-dithiobis(2-nitrobenzoic acid) the hydrolysis of the thiol ester by trypsin-like enzymes provides a continuous colorimetric assay with a sensitivity comparable to the best fluorometric substrates. Z-Lys-SBzl is readily synthesized in good yield, is water soluble, and has a low rate of spontaneous hydrolysis even at pH 8.0. This assay procedure has been routinely used with urokinase, human urinary and human plasma kallikrein, thrombin, plasmin, β-trypsin, factor Xa, and crotalase. Levels of detection of these enzymes are in the range 10−14 to 10−13 mol.