Measurement of the Rate of Plasmin Action on Synthetic Substrates by Phyllis

It has been shown by Troll et al. (1) that plasmin (present in the blood in an inactive form) can hydrolyze esters of arginine and lysine. These authors used a form01 titration method to follow the action of plasmin on TAME (p-toluenesulfonyl-L-arginine methyl ester) and several precipitation and titration methods (2) to follow the action of the enzyme on LME (L-lysine methyl ester). They also tried the Hestrin method (3), which is a calorimetric method based on the formation of a ferric complex of the hydroxamic acid resulting from the reaction of an ester with alkaline hydroxylamine. They found that this procedure gave the same results as the titration methods, but they gave no experimental details and stated that the titration method was preferable because it could be used at optimal substrate concentrations. In this laboratory, we have found that the titration methods, expecially for LME, give poor reproducibility, primarily because of difficult end points. We have modified the Hestrin method so that enzyme rates at optimal substrate concentrations of either TAME or LME can be determined by using the same standard reagents and procedures. With this procedure we find that the rate measurements are reproducible with an average deviation of 3 per cent of the rate.