The Purification of Human Prothrombin^[*]

Summary

A method has been presented for the preparation of human prothrombin. The product is apparently homogenous by chromatographic, ultracentrifugal, electrophoretic and immunochemical analysis. It contains less than 1 part in 10⁷ of thrombin and has a specific activity of 1560 u/mg dry weight. Problems related to the variability in specific activity of prothrombin preparations reported in the literature have been critically reviewed.

^* This investigation was supported in part by Research Grants from the National Science Foundation and National Institutes of Health and was carried out during tenure of National Heart Institute Special Fellowship No. 2-F3-HE-13, 942 (S. S. S.).

^** Present address: Jefferson Medical College, Cardeza Foundation for Hematologic Research, 1015 Sansom Street, Philadelphia, Pa.

• References

• 1 Seegers W. H, Loomis E. G, Vandenbelt J. M. Preparation of prothrombin Products: isolation of prothrombin and its properties. Arch. Biochim 6: 85 1945;
• 2 Seegers W. H. Purification of prothrombin. Record Chem. Progr 13: 143 1952;
• 3 Seegers W. H, Alkjaersig N. Comparative properties of purified human and bovine prothrombin. Amer. J. Physiol 172: 731 1953;
• 4 Lewis M. L, Ware A. G. Simple procedure for separation of prothrombin and accelerator globulin from citrated human plasma. Proc. Soc. exp. Biol. (N. Y.) 84: 636 1953;
• 5 Lanchantin G. F. An analysis of human prothrombin by starch block electrophoresis. Amer. J. Physiol 194: 7 1958;
• 6 Goldstein R, Le Bollock A, Alexander B, Zonderman E. Preparation and properties of prothrombin. J. biol. Chem 234: 2857 1959;
• 7 Asada T, Masaki Y, Kitihara K, Nagayania R, Hatashita T, Yanagisawa I. Conversion of prothrombin into thrombin. J. Biochem 49: 721 1961;
• 8 Lanchantin G. F, Friedmann J. A, DeGroot J, Mehl J. W. Preparation of human plasma prothrombin and some of its sedimentation properties. J. biol. Chem 238: 238 1963;
• 9 Lanchantin G. F, Friedmann J. A. Isolation of human plasma prothrombin of high specific activity by gel filtration. Proc. Soc. exp. Biol. (N. Y.) 114: 584 1963;
• 10 Miller K. D. Chromatographic isolation of plasma prothrombin and trans-a-glucosylase. J. biol. Chem 231: 987 1958;
• 11 Shapiro S. S, Waugh D. F. Purification of human prothrombin. Fed. Proc 23: 459 1964;
• 12 Weiss H. J, Eichelberger Jr. J. W, Crosby W. M. Studies on the differences in activity of lung and brain thromboplastin. J. clin. Invest 40: 205 1961;
• 13 Weiss H. J, Eichelberger Jr. J. W. The detection of platelet defects in patients with mild bleeding disorders. Amer. J. Med 32: 872 1962;
• 14 Flodin P. Dextran gels and their application in gel filtration. Thesis Uppsala (1962)
• 15 Crestfield A. M, Stein W. H, Moore S. On the preparation of bovine pancreatic ribo-nuclease A. J. biol. Chem 238: 618 1963;
• 16 Smithies O. An improved procedure for starch-gel electrophoresis: further variations in the serum proteins of normal individuals. Biochem. J 71: 585 1959;
• 17 Raymond S, Weintraub L. Acrylamide gel as a supporting medium for zone electrophoresis. Science 130: 711 1959;
• 18 Poidik M. D. Starch gel electrophoresis in a discontinuous system fo buffers. Nature (Lond.) 180: 1477 1957;
• 19 Strutt J. W. On the scattering of light by small particles. Philosophical Mag. (4) 41: 447 1871;
• 20 Lowry O. H, Rosebrough N. J, Farr A. L, Randall R. J. Protein measurement with the folin phenol reagent. J. biol. Chem 193: 265 1951;
• 21 Ware A. G, Seegers W. H. Two-stage procedure for the quantitative determination of prothrombin concentration. Amer. J. clin. Path 19: 471 1949;
• 22 Loomis E. C, Seegers W. H. Purified prothrombin: factors which influence its activation. Arch. Biochem 5: 265 1944;
• 23 Whitaker J. R. Determination of molecular weights of proteins by gel filtration on sephadex. Anal. Chem 35: 1950 1963;
• 24 Lamy F, Waugh D. F. Certain physical properties of bovine prothrombin. J. biol. Chem 203 (Suppl. 04) 89 1953;
• 25 Moore S, Stein W. H. Column chromatography of peptides and proteins. Advanc. Protein Chem 11: 191 1956;
• 26 Morris C. J. O. R, Morris P. Separation Methods in Biochemistry. 309 Interscience; New York: 1963
• 27 Mammen E. F, Thomas W. R, Seegers W. H. Activation of purified prothrombin to auto-prothrombin I or autoprothrombin II. (Platelet cofactor II or autoprothrombin II-A). Thrombos. Diathes. haemorrh. (Stuttg.) 5: 218 1961;
• 28 Warner E. D, Brinkhous K. M. Smith Quantitative study on blood clotting: prothrombin fluctuations under experimental conditions. Amer. J. Physiol 114: 667 1936;
• 29 Wagner R. H, Graham J. B, Penick G. D, Brinkhous K. M. In: The Coagulation of Blood: Methods of Study. 105 Tocantins L. M. editor Grune and Stratton; New York: 1955
• 30 Shulman N. R, Hearon J. Z. Kinetics of conversion of prothrombin to thrombin by biological activators. J. biol. Chem 238: 155 1963;
• 31 Minimum Requirements for Dried Thrombin, Second Revision, Division of Biologies Standards, National Institutes of Health, Bethesda, Maryland 1946.
• 32 Waugh D. F, Baughman D. J, Miller K. D. In: The Enzymes. Boyer P. D, Lardy M, Myrback K. (eds.) 2nd ed.. vol. 4 215 Academic Press; New York: 1960