The action of thrombin on peptide p-nitroanilide substrates: hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human alpha and gamma and bovine alpha and beta-thrombins.

Human and bovine alpha-thrombins (greater than 90% alpha form) with high fibrinogen clotting activities (approximately 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 degrees C, pH 7.8 are: (Km = 4.18 +/- 0.22 and 3.61 +/- 0.15 microM; kcat = 127 +/- 8 and 100 +/- 1 s-1) for Chromozym TH and (Km = 1.33 +/- 0.07 and 1.50 +/- 0.10 microM; kcat = 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 gamma-thrombin (approximately 5 units/mg) and bovine beta-thrombin (approximately 200 units/mg), give increased values for these parameters (km = 14.3 +/- 2.4 and 14.4 +/- 2.2 microM; kcat = 160 +/- 9 and 124 +/- 6 s-1) for Chromozym-TH; and (Km = 2.50 +/- 0.36 and 2.99 +/- 0.33 microM; kcat = 106 +/- 3 and 106 +/- 3 s-1) for S-2238. Based on these parameters, 50% degradation of human or bovine alpha-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 alpha forms are erroneously used and assays are at 150 microM 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 less than 0.1 nM or 0.01 clotting unit/ml of alpha-thrombin.