Human alpha- and gamma-thrombin specificity with tripeptide p-nitroanalide substrates under physiologically relevant...

Kinetic parameters [the Michaelis-Menten (Km), catalytic (kcat), and specificity (kcat/Km) constants] were determined for human alpha- and gamma-thrombins with the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-p-nitroanilide), Chromozym-TH (Tos-Gly-Pro-Arg-p-nitroanilide), and Spectrozyme-TH (H-D-HHT-Ala-Arg-p-nitroanilide) under physiologically relevant conditions (0.15 M NaCl buffered with 10 mM HEPES and 10 mM Tris-HCL, pH 7.4 at 37 degrees C). No major differences were found between alpha-thrombin with high fibrinogen clotting activities (greater than 3,500 killo clotting units/g) and gamma-thrombin with essentially no clotting activities (less than 10 kCU/g), although the Km values and in most cases kcat values for alpha-thrombin were slightly lower than for the gamma-thrombin. At 37 degrees C, relative to 23 degrees C, Km values increased 2-fold for S-2238, approximately 1.5-fold for Chromozym-TH, and remained essentially the same for Spectrozyme-TH (e.g., reciprocal substrate binding), whereas the kcat values increased for all 3 substrates (e.g., enzyme turnover). This caused kcat/Km values to decrease slightly for S-2238, remain the same for Chromozym-TH, and increase for Spectrozyme-TH (e.g., enzyme efficiency). Since spontaneous hydrolysis was not limiting at 37 degrees C, assays employing these substrates may be satisfactorily performed under physiologically relevant conditions. Under these conditions, kcat/Km ratios for the 3 substrates are similar to that for the A alpha cleavage in fibrinogen by alpha-thrombin.