Isolation and Purification of Prothrombin from Dicumarolized Steers

Prothrombin was isolated and highly purified from the plasma of steers treated with Dicumarol when their plasma prothrombin concentration approximated 12% of normal. The preparations showed a single component by ultracentrifugation, immunodiffusion, immunoelectrophoresis, polyacrylamide (disc) gel electrophoresis using different separatory gel concentrations with running gel at pH 7.5 and 8.9, and also by moving boundary electrophoresis at pH 7.0 and 8.6. Preparations did not undergo dissociation or inactivation during moving boundary electrophoresis. The specific activity of the preparations was 1800 ± 300 units per mg of protein, approximately half that of normal prothrombin (3200 ± 200 units per mg of protein). Prothrombin purified from plasma at a concentration of 50% of normal had normal specific activity, however. Prothrombin prepared from normal plasma diluted to a prothrombin concentration of 12.5% of normal had specific activity similar to those preparations from normal undiluted plasma, but greater than that from Dicumarolized plasma. The low specific activity of the Dicumarolized prothrombin is considered indicative of the biosynthesis of altered prothrombin molecules.