The Purification of Human Prothrombin

S. S Shapiro; D. F Waugh

doi:10.1055/s-0038-1655603

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1966; 16(03/04): 469-490
DOI: 10.1055/s-0038-1655603

Originalarbeiten — Original Articles — Travaux Originaux

Schattauer GmbH

The Purification of Human Prothrombin^[*]

S. S Shapiro^**

¹Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts

,

D. F Waugh

¹Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts

› Author Affiliations

Abstract

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.

Full Text

References

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

The Purification of Human Prothrombin[*]

The Purification of Human Prothrombin^[*]