Preparation of Highly Purified Prothrombin Complex: I. CRYSTALIZATION, BIOLOGICAL ACTIVITY, AND MOLECULAR PROPERTIES

Abstract

Abstract Bovine prothrombin, an α2-glycoprotein clotting factor of plasma, has been isolated in high degree of purity in crystalline form as the barium glycoprotein product of interaction. Chemical determinations made on the product showed the following composition, in percentage of the dry weight: protein, 21.5%; barium, 32%; and citrate, 8.9%. That the crystals represent a true prothrombin-metal complex has been established by control studies, x-ray diffraction pattern, chemical composition, and recrystallization. The product showed constant solubility characteristics. Extensive physical and chemical studies of the barium-free protein are presented. Further purification was achieved by gel filtration on Sephadex G-100. The peak fractions evidenced activity of 3000 Iowa units (Seegers, W. H., Prothrombin, Harvard University Press, Cambridge, Massachusetts, 1962, p. 420) per mg (corresponding to a 600-fold purification), as well as appreciable Factors VII, IX, and X coagulant activities. This product is designated prothrombin in accordance with the nomenclature used by other investigators. The preparation was homogeneous by sedimentation velocity pattern, sedimentation equilibrium analysis, and gel filtration. Despite these indications of homogeneity, physical heterogeneity was shown by immuno- and disc electrophoresis. The multiple coagulant activities and protein subcomponents were found to be homogeneous with respect to molecular weight (70,477 ± 2,789), determined by sedimentation equilibrium in a dissociating solvent consisting of 6 m guanidine hydrochloride and 0.5% mercaptoethanol, and also with respect to Stokes radius. Physical constants, determined in various systems, include diffusion constant, sedimentation coefficient, frictional ratio, isoelectric point, isoionic point, electrophoretic mobility, and extinction coefficient. Physical data failed to show evidence of dissociation into subunits or fragmentation by dilution to below 0.1% protein or by prolonged exposure to a dissociating solvent. The latter observation suggests that the reduction in sedimentation constant noted in high ionic strength solvents in this study, and by other observers, is probably attributable to changes in shape and hydration of the kinetic unit. The product was also shown to undergo rapid, reversible association in solvents of low ionic strength (T/2 = 0.15). DEAE-Sephadex chromatography resulted in partial activation of the complex, and several homogeneous protein species were isolated and partially characterized. The monomeric molecular weights determined by sedimentation equilibrium in a solvent of 6 m guanidine hydrochloride and mercaptoethanol were as follows: (devoid of Factors VII and X), 65,530 ± 1,247; modified zymogen, 52,395 ± 3,449; Factor VII, 33,900 ± 3,390; and Factor X, 37,772 ± 1,234. The modified zymogen was inert in the two-stage assay and in 25% sodium citrate, but readily transformed to thrombin when Factor X was added. The results are consistent with the interpretation that the complex, as isolated in this study, comprises a family of glycoproteins that can undergo a proenzyme-enzyme transformation and that have similar molecular properties but separate clotting activities.