抄録
Canine prothrombin was purified through following procedures: Combination of BaSO4 adsorption, elution of adsorbed prothrombin in 0.25M sodium citrate, concentration of the eluate by (NH4)SO4 fractionation at 80% saturation and DEAE-cellulose chromatography.
The prothrombin preparation thus obtained was proved to be pure by immunoelectrophoresis but some trace impurities were visible by SDS polyacrylamide gel electrophoresis (SDS PAGE).
The molecular weight was 81, 300±850 (SD) on SDS PAGE, the extinction coefficient was 13.8±0.4, and the specific activity was 1, 200±50 NIH units per mg prothrombin.
Then, the activation behavior of prothrombin by activated Factor X, in the presence of Factor V, phospholipids and calcium was determined by SDS PAGE. The molecular weights for P2, Fa, P3 and Fb were 60, 000, 28, 540, 40, 400 and 21, 240 respectively.
Next, thrombin was separated by using QAE-Sephadex A50 chromatography. The molecular weight of thrombin averaged 38, 111±500, the extinction coefficient was 16.0±0.2 and the specific activity was 2, 000±65 NIH units per mg thrombin. One peculiar finding with the canine material was that P2 appeared to be double band in SDS PAGE. Further studies are required to elucidate its significance.
