Thrombokinase has been isolated from bovine plasma by a procedure which begins with the highly purified product of a previously described method, chromatographs it on DEAE-cellulose, and then fractionates it by continuous flow electrophoresis, yielding 0.2 mg per liter of oxalated plasma. The electrophoretic fraction has shown a single boundary in the ultracentrifuge; and its esterase activity on toluenesulfonylarginine methyl ester has been about the same as that of thrombokinase previously isolated by repeated electrophoretic fractionations. Thrombokinase is a euglobulin with minimum solubility near pH 5.0. It is most stable within the pH range 7.5 to 9.5; but there is also a peak in the stability curve near pH 1.8. A few micrograms of thrombokinase per milliliter can activate prothrombin in the presence of EDTA. A few thousandths of a microgram causes rapid production of thrombin in the system: prothrombin, thrombokinase, calcium chloride, phosphatide, "accelerator." But, thrombokinase has less than 1/175 the proteolytic activity of crystallized trypsin.Thrombokinase was first isolated from bovine plasma by a series of three electrophoretic fractionations (1). Later, it was reported (2, 3) that chromatography on DEAE-cellulose offered a less arduous means for attaining a comparable specific activity. However, it has been found that the chromatographic preparation is not homogeneous electrophoretically. Now, one chromatographic step and one electrophoretic step have been incorporated into the procedure for isolation of thrombokinase.Thrombokinase so prepared, has about the same esterase activity on toluenesulfonylarginine methyl ester (TAMe) as thrombokinase prepared by the more laborious procedure; and it sediments in the ultracentrifuge with a single, grossly symmetrical boundary. A few of its outstanding enzymatic and physicochemical properties are herein delineated.
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