Evaluation of Disulfide Bonds and Sulfhydryl Groups in the Blood Clotting Mechanism

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Abstract
Employing 2-stage clotting systems, it was observed that compounds commonly used to alter the disulfide linkage of proteins, in appropriate concentrations, inhibited coagulation by decreasing the reactivity of assayable purified prothrombin; those used to alter sulfhydryl groups had no effect on clotting. Reversal of inhibition was accomplished both by dialysis and by oxidation. Using the argentimetric ampero-metric method, no -SH was detected in purified prothrombin, thrombin or fibrinogen, nor was any detectable -SH evolved during the conversion of prothrombin to thrombin. By successfully adapting the amperometric method to the quantitative determination of disulfides in intact proteins, -S-S- was measurable in purified prothrombin (4M-S-S-/M) and thromboplastin, but in thrombin and fibrinogen only if denatured. By simultaneously determining and correlating -SH concentration and prothrombin activity, variables, such as oxidation phenomena and protein concentration in the clotting systems, were shown to exert a profound effect on prothrombin inhibition by -S-S- antagonists. The data indicate that disulfide bonds are present in and probably essential for the biological activity of, prothrombin.