Separation of human factor X from factor Xa by reversed-phase high-performance liquid chromatography

Abstract

Human Factor X is the vitamin K-dependent proenzyme of a plasma serine protease that participates in the cascade of events leading to blood coagulation. It is converted to its active form, factor Xa, after specific cleavage by other plasma proteases or the protease from Russel's Viper venom. We have separated Factor X from factor Xa by reversed-phase high-performance liquid chromatography using an increasing gradient of acetonitrile in 0.1% trifluoroacetic acid. The factor X and factor Xa activities were well separated from each other on a wide-pore diphenyl column (Whatman Protesil 300) in less than 30 min. Both factor X and factor Xa activities were found to be essentially unaffected by the solvent system. This system was used to evaluate the purity of several factor X and factor Xa preparations. The kinetics of the Russel's Viper venom catalyzed conversion of factor X to factor Xa was also studied by using this chromatography system. A time-dependent decrease in the protein peak corresponding to factor X and a corresponding increase in the factor Xa protein peak was observed upon incubation with Russel's Viper venom.