Activation of bovine Factor X by the venom coagulant protein of Vipera russelli: Complex formation of the activation fragments

Abstract

The interaction of the fragments of Factor X which are formed during the activation of Factor X by the coagulant protein of Russell's viper venom has been examined. Factor X and activated Factor X yielded single protein peaks with identical elution volumes by gel filtration on columns of Sephadex G-75 or BioGel P-100 equilibrated in 0.02 M Tris-HCl, 0.1 M NaCl, pH 7.4, or 8 M urea. However, an activation peptide with a molecular weight of 11 000 could be separated from a higher molecular weight activation fragment by gel filtration on columns of BioGel P-100 equilibrated in 1% sodium dodecylsulfate and 8 M urea. This activation peptide was also observed by gel electrophoresis in sodium dodecylsulfate and urea using highly cross-linked polyacrylamide gels. The molecular weight and composition of the activated Factor X complex, prepared by gel filtration in 8 M urea, were compared to those of Factor X. Molecular weights, determined by equilibrium centrifugation in 6 M guanidine·HCl, are 56 000 and 55 000 for Factor X and the activated Factor X complex, respectively. The amino acid compositions were indistinguishable; slight differences in carbohydrate content were observed. It appears that Factor X undergoes proteolytic cleavage of its heavy subunit during activation by the coagulant protein and a complex of the two activation fragments of Factor X are formed. The binding of these activation fragments in the presence of Tris-HCl, pH 7.4, 8 M urea, or 6 M guanidine·HCl to form an activated Factor X complex suggests the high degree of complementarity and mutual affinity of these polypeptide chains.