Thrombin specificity with tripeptide chromogenic substrates: Comparison of human and bovine thrombins with and without fibrinogen clotting activities

Abstract

To assess the thrombin specificity of tripeptide chromogenic substrates, we determined the Michaelis-Menten (K(m)), catalytic (k(cat)), and specificity (k(cat)/k(m)) constants for S-2238 (H-D-Phe-Pip-Arg-p-nitroanilide), chromozym-TH (Tos-Gly-Pro-Arg-p-nitroanilide), and spectrozyme-TH (H-D-hexahydrotyrosyl-Ala-Arg-p-nitroanilide) with high-purity thrombin preparations. Human and bovine α-thrombins, prepared by essentially the same procedure, were each >95% in the form of the enzyme (α-thrombin) and had a specific fibrinogen-clotting activity > 2000 kilo-clotting units per gram of protein (kcu/g). In contrast, human γ- and bovine β-thrombins, made by controlled passage of α-thrombin through trypsin agarose, were >98% of their respective forms and had fibrinogen-clotting activity < 1 kcu/g. With the tripeptide chromogenic substrates these four thrombin forms at pH 7.8 and 23°C had K(m) values of 1.6 to 16 mol/L, K(cat) values of 35 to 130 s-1, and K(cat)/K(m) ratios of 4.7 to 52 L.mol.s-1. Although K(m) for an individual substrate was slightly higher for bovine than for human α-thrombins and although K(m) values for the nonclotting forms (human γ- and β-thrombins) were higher than for clotting forms (α-thrombins), we found no major differences among the kinetic values for the three substrates.