The effect of the initial pH and the concentrations of thrombin, fibrinogen, and Ca2+ upon the rate of pH change associated with clotting of bovine fibrinogen by human thrombin was investigated at pH 6.80, 7.80, and 8.80, 0.3 ionic strength, 25-degrees-C, and 19.5 mg/mL final fibrinogen concentration. At pH 6.80 and 7.80, the reaction was first order, with rate constant k1. At pH 8.80, a first-order reaction of the release of H+ (k1) was followed by a partial rebinding of these in a reaction consecutive to the first one (k2). At each of the above pH values, k1 was proportional to thrombin concentration in the 0.05-3.0 min-1 range investigated. The k1 constants were 0.111+/- 0.001, 0.250 +/- 0.005, and 0.190 +/- 0.002 min-1 (NIH thrombin units)-1 mL-1 at pH 6.80, 7.80, and 8.80, respectively. Plots of log rate vs log thrombin concentration of these data were linear with slopes close to 1 at all three pH values. The rate of the second reaction (k2) was independent of both the thrombin and the initial fibrinogen concentration. The pH dependence of k1 exhibited a bell-shaped curve that could be resolved into the effect of one group with a pK of 7.27 that increased the rate and another with a pK of 9.22 that decreased the rate. With constant thrombin concentration but varying fibrinogen concentration, plots of 1/k1 vs [fibrinogen] were linear, but the lines did not pass through the origin. From the slope and intercept, k(cat) and K(M) of the Michaelis-Menten equation could be calculated. The same parameters were obtained also from initial velocity vs [fibrinogen] plots. Values of k(cat) were consistent and accurate; those of K(M) were more scattered. K(M) was (22.4-34.2) X 10(-6) M at pH 6.80 and approximately 7 X 10(-6) M in the pH 7.26-8.80 range. The latter value, pertaining to the release of H+ ions, is in agreement with values in the literature for K(M) of the release of fibinopeptide A by thrombin in the 7.4-8.0 pH range. The value of k(cat) s-1 (unit of thrombin)-1 mL-1 increases from 1.2 X 10(-10) s-1 unit of thrombin-1 mL-1 at pH 6.80 to 2.46 X 10(-10) at pH 7.80 and then decreases to 2.01 X 10(-10) 10(-1) (units of thrombin)-1 mL-1 at pH 8.80. The k(cat) values are significantly lower than those in the literature for the release of fibrinopeptide A. The difference may be caused by the higher ionic strength (0.3 vs 0.15). The amounts of H+ liberated were 3.0, 1.9, and 3.0 equiv of H+ mol of fibrinogen at the respective pH values. The pH shift was correlated with the release of fibrinopeptides and with polymerization, the latter monitored by the increase of turbidity. Release of fibrinopeptide A followed first-order kinetics; release of fibrinopeptide B followed a first-order reaction consecutive to release of fibrinopeptide A. At pH 6.80, the pH shift lagged behind the release of FPA, by a factor of 0.6. Most of the H+ at this pH originates in the polymerization or changes following this. Therefore, the delay must occur in this phase. At pH 7.80, pH shift and release of FPA are simultaneous. There is no indication of contribution from release of FPB to the pH shift. The stoichiometry of H+ released also supports this contention. The most likely explanation of this finding is an increase by the polymerization reaction of the pK of the alpha-NH2 groups formed by release of FPB. At pH 8. 80, the pH shift again lags behind release of FPB; however, the reaction curve can be reconstructed accurately by assuming that FPA contributes fully, while FPB to about half the extent, to the pH shift. This places the pK of the groups produced by release of the latter to approximately 8.8. At all three pH values, the turbidity curves coincided or were slightly ahead of the FPA release curves, showing that the latter have a determining role in polymerization and FPB is not directly involved. The pH shift and release of fibrinopeptides was also investigated with bovine thrombin on bovine fibrinogen at the above 3 pH values. Significant differences were found between the human and the bovine enzyme.
