Preparation of human plasma prothrombin and some of its sedimentation properties.

Although there have been a number of studies on the physical properties of bovine prothrombin (2-5) and, to some extent, equine prothrombin (6), there are yet few data available on preparations obtained from human plasma. This is largely due to the fact that methods customarily employed to isolate prothrombin from bovine plasma, when applied to human plasma, inevitably result in products of inferior physicochemical purity (7-10). Prothrombin preparations of high specific activity, isolated from human plasma by the method of Seegers, have been found to be heterogeneous in the ultracentrifuge (7), and it has been suggested that their specific activity might be twice as great as that of the bovine product (8). In one published experiment, Alexander (10) has shown that a highly purified human preparation isolated by his method (9) was heterogeneous by sedimentation velocity analysis, although 90% of the product had a sedimentation coefficient similar to that of bovine prothrombin. Prothrombin, isolated from human acid-citrate-dextrose Solution B plasma by the Lewis and Ware method (11) is also polydispersed in the ultracentrifuge (12). Modifying the Seegers method to include chromatography on diethylaminoethyl cellulose, Asada et al. (13) prepared a human product that sedimented as a single component in the ultracentrifuge; however, the period of analysis reported was only 16 minutes. Because there appear to be a number of bovine prothrombin derivatives with biological properties similar to certain of the human coagulation factors necessary for the conversion of prothrombin to thrombin (14, 15), a more detailed analysis of the physical properties of human prothrombin appears warranted. In this communication, we have modified the Lewis and Ware procedure (11) for the isolation of prothrombin from human acid-citrate-dextrose Solution B plasma. This modification was found to be necessary since, although this procedure results in preparations of high specific activity and yield, the products obtained are not homogeneous by starch block electrophoresis or sedimentation velocity analysis (12) and contain at least six components by immunoelectrophoresis (1). Our procedure included the batchwise adsorption on diethylaminoethyl cellulose of a redissolved barium citrate eluate of plasma containing prothrombin, with the successive washing of the resin to remove other proteins and the final elution of the zymogen in sodium