Simultaneous Purification of Bovine Prothrombin and Factor X: ACTIVATION OF PROTHROMBIN BY TRYPSIN-ACTIVATED FACTOR X

Abstract

Abstract A simple reproducible procedure has been developed for the large scale isolation of bovine factor X and prothrombin in highly purified states. Both proteins isolated by this method show a single component in four different electrophoretic systems. The specific activity of prothrombin obtained by this procedure is 1300 ± 100 NIH thrombin units per mg of prothrombin, whereas the specific activity of factor X is 90 ± 10 units of activated factor X (factor Xa) per mg of factor X, when assayed by the standard technique (Bachmann, F., Duckert, F., and Koller, F. (1958) Thromb. Diath. Haemorrh. 2, 24). The over-all yields of bovine prothrombin and factor X are 70 mg and 4.5 mg, respectively, per liter of starting plasma. The isolation procedure involves adsorption of factor X and prothrombin on to barium citrate, their elution from the barium citrate complex with EDTA, extensive dialysis against 0.02 m trisodium citrate and 0.9% sodium chloride, ammonium sulfate fractionation, diisopropylfluorophosphate treatment, and DEAE-cellulose chromatography. DEAE-cellulose chromatography in (sodium) citrate buffer at pH 6.0 effectively and completely separates prothrombin from factor X. Factor X is then further purified by DEAE-Sephadex chromatography. Both proteins are completely stable in 50% glycerol-H2O (v/v) at -20° for at least 18 months. The molecular weights of bovine prothrombin and factor X as determined by sodium dodecyl sulfate polyacrylamide electrophoresis are 72,000 ± 4,000 and 56,000 ± 2,000, respectively. The molecular weight of native factor X, determined by sedimentation equilibrium, is 59,000 ± 2,000. Factor X protein is composed of two chains of 44,000 and 15,500 daltons as determined by gel filtration of the reduced and S-carboxymethylated protein in 6 m guanidinium chloride. Activation of factor X by trypsin insolubilized on polyacrylamide produced a trypsin-free factor Xa(factor Xa(trypsin)) with a maximum yield of 750 ± 50 units of factor Xa per mg of factor X, which is 8 times higher than the yield obtainable by the action of Russell's viper venom (RVV) on factor X. Activation of prothrombin by factor Xa(trypsin), has been carried out under conditions in which the possibility of thrombin contamination has been eliminated. The rate of the factor Xa(trypsin) catalyzed activation of prothrombin is accelerated 50-fold in the presence of Ca2+ and 90-fold in the presence of both Ca2+ and phospholipid. However, under all conditions studied, and all types of factor Xa (factor Xa(trypsin, RVV, citrate)) used, the products of prothrombin activation are the same and electrophoretically equivalent to those described previously by this laboratory (Mann, K. G., Heldebrant, C. M., and Fass, D. N. (1971) J. Biol. Chem. 246, 6106).