Isolation of the high molecular form of bovine factor X and some of its physical properties

Abstract

A high molecular form of bovine factor X has been isolated from freshly collected bovine blood by BaSO 4 absorption, exhaustive washing with 0.001 M BaCl 2 and chromatographed on DEAE-cellulose column employing a linear salt gradient. This isolated factor X showed a single protein band on analytical polyacrylamide gel disc electrophoresis. Only one single protein peak was observed in the chromatogram of DEAE-Sephadex A-50 chromatography conducted at 3 °C. Sedimentation equilibrium analysis of this bovine factor X revealed no apparent heterogeneity or self association-dissociation phenomena. It yielded a weight-average molecular weight of 74 000 for the native factor X. In the absence of any reducing agent, factor X migrated in dodecyl sulfate gel electrophoresis as a single component with an estimated molecular weight of 74 300. Both dodecyl sulfate gel electrophoresis in the presence of 2-mercaptoethanol and agarose gel chromatography in 6 M guanidinium chloride revealed that this native factor X is composed of two polypeptide chains of molecular weights of 56 000 and 22 100. Factor X can be converted to the enzymatically active factor X a by Russell's viper venom and in the presence of Ca 2+. Factor X a was purified by DEAE-cellulose chromatography. This Russell's viper venom activated factor X a also showed a single protein band upon analytical polyacrylamide gel disc electrophoresis. Sedimentation equilibrium analysis of this factor X a yields a weight-average molecular weight of 59 000 with no apparent heterogeneity or self-association phenomena. In the absence of any reducing agent, factor X a migrated as a single component in dodecyl sulfate gel electrophoresis with an estimated molecular weight of 58 500. From the results of dodecyl sulfate gel electrophoresis in the presence of 2-mercaptoethanol as well as agarose gel chromatography in 6 M guanidinium chloride, factor X a is also composed of two polypeptide chains of molecular weights of 36 700 and 22 800. Therefore, the heavy and light chains of both native factor X and factor X a are linked together by disulfides.