The action of thrombin on peptide p-nitroanilide substrates: Hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human α and γ and bovine α and β-thrombins

Abstract

Human and bovine α-thrombins (>90% a form) with high fibrinogen clotting activities (∼3,000 U.S. units/mg protein) exhibit similar Michaelis Menten kinetics with the p-nitroanilide tripeptide substrates Tos-Gly-Pro-Arg-pNA (Chromozym-TH) and D-Phe-Pip-Arg-pNA (S-2238). The kinetic parameters at I = 0.11 M, 25°C, pH 7.8 are: (K m = 4.18 ± 0.22 and 3.61 ± 0.15 μM; k cat = 127 ± 8 and 100 ± 1 s −1) for Chromozym TH and (K m = 1.33 ± 0.07 and 1.58 ± 0.10 μM; k cat = 91.4 ± 1.8 and 98.0 ± 0.5 s −1) for S-2238 for the human and bovine enzymes, respectively. Unlike the native enzyme forms, their “non-clotting” terminal degradative forms, human γ-thrombin (∼5 units/mg) and bovine β-thrombin (∼200 units/mg), give increased values for these parameters (K m = 14.3 ± 2.4 and 14.4 ± 2.2 μM; k cat = 160 ± 9 and 124 ± 6 s −1) for Chromozym-TH; and (K m = 2.50 ± 0.36 and 2.99 ± 0.33 μM; k cat = 106 ± 3 and 106 ± 3 s −1) for S-2238. Based on these parameters, 50% degradation of human or bovine α-thrombins can be calculated to produce relatively small errors in the kinetic measurement of total thrombin concentrations (maximally 9% and 7% for Chromozym-TH; 7% and 3% for S-2238, respectively) if the kinetic parameters for all a forms are erroneously used and assays are at 150 μM substrate. This is in contrast to the large errors inherent in clotting activity measurements on thrombin mixtures. Incorporation of 1 mg/ml of polyethylene glycol 6,000 into assay solutions eliminates systematic errors otherwise caused by thrombin adsorption to surfaces and enables thrombin to be accurately assayed at concentrations <0.1 μM or 0.01 clotting unit/ml of α-thrombin.