Methods for the Concentration of Bovine Ac-Globulin (Factor V)^[*]

Summary

An attempt was made to obtain Ac-globulin from bovine plasma. The concentrates contain mostly protein, and phosphorus is also present. The stability characteristics vary from one preparation to another, but in general there was no loss before 1 month in a deep freeze or before 1 week in an icebox, or before 5 hours at room temperature. Reducing agents destroy the activity rapidly. S-acetylmercaptosuccinic anhydride is an effective stabilizing agent. Greatest stability was at pH 6.0.

In the purification bovine plasma is adsorbed with barium carbonate and diluted 6-fold with water. Protein is removed at pH 6.0 and the Ac-globulin is precipitated at pH 5.0. Rivanol and alcohol fractionation is followed by chromatography on Amberlite IRC-50 or DEAE-cellulose. The final product is obtained by isoelectric precipitation.

^* This investigation was supported by a research grant H-3424 from the National Heart Institute, National Institutes of Health, U. S. Public Health Service.

• References

• (1) Ware A. G, Murphy R. C, Seegers W. H. The Function of Ac-globulin in Blood Clotting. Science 106: 618 1947;
• (2) Landaburu R. H, Seegers W. H. The Acetylation of Prothrombin. Canad. J. Biochem. 38: 613 1960;
• (3) Seegers W. H, Landaburu R. H. Acceleration of Thrombin Activity with Ac-globulin. Amer. J. Physiol. in press
• (4) Owren P. A. The Coagulation of Blood, Investigations on a New Clotting Factor. Acta med. scand. Suppl. 194 1947;
• (5) Ware A. G, Seegers W. H. Plasma Accelerator Globulin: Partial Purification, Quantitative Determination, and Properties. J. biol. Chem. 172: 699 1948;
• (6) Owren P. A. The Fifth Coagulation Factor (Factor V) Preparation. Biochem J 43: 136 1948;
• (7) Lewis M. L, Ware A. G. A Simple Procedure for Separation of Prothrombin and Accelerator Globulin from Citrated Human Plasma. Proc. Soc. exp. Biol (N Y ) 84: 636 1953;
• (8) Cox F. M, Lanchantin G. F, Ware A. G. Chromatographic Purification of Human Serum Accelerator Globulin. J. clin. Invest. 35: 106 1956;
• (9) Fenichel R. L, Rose L. An Investigation of the Properties of Ac-globulin Prepared by a New Isolation Technique. Thromb. Diath. haem. 5: 601 1961;
• (10) Surgenor D. M, Steele B. B, Wallach D. F. H. Thrombin Formation in Purified Systems. Thromb. Diath. haem. 3: 214 1959;
• (11) Wolfson W. Q, Cohn C, Calvary E, Ichiba F. Rapid Procedure for the Estimation of Total Protein, True Albumin, Total Globulin, Beta Globulin and Gamma Globulin in 1.0 ml of serum. Amer. J. clin. Path. 18: 730 1948;
• (12) Horwitt B. N. Determination of Inorganic Serum Phosphate by Means of Stannous Chloride. J. biol. Chem. 199: 537 1952;
• (13) Seaman A. J, Owren P. A. An Asolectin Adsorbed Substrate for Proaccelerin Assay. J. clin. Invest. 35: 145 1956;
• (14) Ware A. G, Seegers W. H. Serum Ac-globulin: Formation from Plasma Ac-globulin; Role in Blood Coagulation; Partial Purification; Properties; and Quantitative Determination. Amer. J. Physiol. 152: 567 1948;
• (15) Landaburu R. H, Seegers W. H. Activation of Prothrombin. Amer. J. Physiol. 193: 169 1958;