Optimization of Enzyme-Based Assays in Coagulation Testing

Optimized assays for antithrombin ill and plasminogen have been developed based on a study of the kinetic parameters Km and I#{231}for four commercially available substrates: the p-nitroanilide derivatives of D-Phe-pipecolyl-Arg (S-2238), and toluenesulfonyl-Gly-Pro-Arg (Chromozym#{174}TH), which are thrombin substrates; o-Val-Leu-Lys (S-2251), a plasmino-gen/streptokinase substrate; and a-N-carbobenzoxy-L-iysine thiobenzyl ester, a substrate for both enzymes. We used a centrifugal analyzer system for rapid data acquisition and interactive analysis. Optimized conditions for assay of a particular enzyme are not constant for different substrates in the same buffering agent. For example, in 1,4-piperazine diethanesulfonic acid buffer at 37 #{176}C,thrombin-catalyzed hydrolysis of Chromozym TH is optimal at 125 mmol/L buffer, 100 mmol/L NaCI, and pH 8.2, whereas substitution of S