The Purification and Properties of Activated Factor X

Abstract

Abstract Partially purified bovine plasma Factor X was converted to its actively enzymatic form by incubation with Russell's viper venom. The activation product was purified by chromatography on diethylaminoethyl cellulose using a gradient 0.09 m to 0.15 m citrate buffer system. A low molecular weight contaminant was removed by dialysis. Activated Factor X isolated in this manner showed a single peak in the analytical ultracentrifuge. The slope of s20,w versus concentration was positive with s020,w = 3.90, while the slope of D020,w versus concentration was negative with D020,w = 7.02 x 10-7. With v = 0.715, obtained from the amino acid analysis, the molecular weight calculated from the Svedberg equation was 47,900 ± 1,000. Low speed sedimentation equilibrium data gave plots of 1n c versus r2 showing upward curvature with a best fit Mw = 47,000 ± 2,000. High speed meniscus depletion runs gave Mw = 47,800 ± 1,000 calculated from the linear portion of the In c versus r2 plots. Chromatography on Sephadex G-200 indicated that a single protein was present (estimated molecular weight 48,000) showing a distinct Gilbert effect. Disc electrophoresis at pH 9.5 gave a single major band but sodium dodecyl sulfate acrylamide gel electrophoresis in the presence of mercaptoethanol gave two bands (estimated molecular weights 20,000 and 30,000). The larger component incorporated 32P after inhibition of the enzymatic activity with [32P]diisopropyl phosphorofluoridate. The amino acid analyses of protein hydrolysates were characterized by large proportions of glutamic and aspartic acids. Two amino-terminal amino acids (alanine and isoleucine) and one carboxyl-terminal (arginine) have been found. Analyses for hexoses, amino sugars, and neuraminic acids were negative. It is concluded that Factor X activated with Russell's viper venom is a protein having a molecular weight about 48,000 and consisting of two polypeptide chains, which undergoes reversible association-dissociation in aqueous solution. These data are clearly at variance with those reported for autoprothrombin c, an enzyme having similar biological activity.