The action of thrombin on peptide p-Nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction condtions

Kinetic parameters for the action of bovine α-thrombin on 24 commercially available peptide p-nitroanilides have been determined. The selectivity constant, k_cat K_m, ranges from 3.3 · 10¹ to 1.1 · 10⁸ M^-1 · s^-1 for the poorest and the best substrates, respectively. The best substrates for thrombin were identified as those with arginine in the P₁ position, proline or a proline homolog in the P₂ position, and an apolar amino acid in the P₃ position. Quantitative distinction between lysine and arginine in the P₁ position and other amino acids in the P₂-P₄ positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effects of NaCl, CaCl₂ and poly(ethylene glycol) concentrations, pH and temperature on the action of thrombin on selected substrates have been assessed. A source of large systematic error in thrombin concentration estimates was identified as resulting from adsorption losses. These losses were eliminated by inclusion of poly(ethylene glycol) in dilution and reaction buffers.