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

A high molecular form of bovine factor X has been isolated from freshly collected bovine blood by BaSO4 absorption, exhaustive washing with 0.001 M BaCl2 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 degrees 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 Xa by Russell's viper venom and in the presence of Ca2+. Factor Xa was purified by DEAE-cellulose chromatography. This Russell's viper venom activated factor Xa also showed a single protein band upon analytical polyacrylamide gel disc electrophoresis. Sedimentation equilibrium analysis of this factor Xa 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 Xa 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 Xa 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 Xa are linked together by disulfides. Great care was taken in washing the BaSO4 precipitate and it is this effective washing which enabled us to isolate the higher molecular from of bovine factor X.