Human Prothrombin: A New Method of Preparation from a Single Individual

Summary

A simple method for purification of human prothrombin from ACT) plasma of one single individual is described. This method is based on three steps :

1. isolation of crude prothrombin complex by barium citrate adsorption;

2. first purification by hydroxyapatite chromatography;

3. further purification by DEAE Sephadex chromatography.

The final product was obtained with a good yield and exhibited less than 5 p. cent contamination. The human purified prothrombin obtained in this way displays :

– one single chain by SDS electrophoresis,

– a molecular weight of 75,000-80,000 daltons,

– a pi value pH 4.4 by electrofocusing,

– Alanine as थ terminal residue.

Amino-acid analysis is reported.

^* Institut d’Université, Groupe U 15 de l’Institut National de la Santé et de la Recherche Médicale, Laboratoire associé au Centre National de la Recherche Scientifique.

• References

• 1 Aronson D. L, and Ménaché D. 1966; Chromatographic analysis of the activation of human Prothrombin with human Thrombokinase. Biochemistry 5: 2635.
• 2 Aronson D. L, Mustafa A. J, and Mushinski J. F. 1969; Purification of human Factor X and comparison of peptide maps of human factor X and Prothrombin. Biochemica et Biophysica Acta 188: 25.
• 3 Bbnamon-Djiane D, Josso F, and Soulier J. P. 1968; Purification de la Prothrombine humaine par Chromatographie sur DEAE Sephadex. Propriétés de la Prothrombine purifiée. Coagulation 3: 259.
• 4 Bencze W, and Schmid K. 1957; Determination of Tyrosine and Tryptophan in Proteins. Analytical Chemistry 29: 1193.
• 5 Davis B. J. 1964; Disc Electrophoresis II: method and application to human serum proteins. Annals of the New York Academy of Sciences 121: 404.
• 6 Finlayson G. R, and Chbambach A. 1971; Isoelectric focusing in Polyacrylamide gel and its preparative application. Analytical Biochemistry 40: 292.
• 7 Goldstein R, and Zonderman E. B. 1960 Purification of Prothrombin and its separation from other coagulation factors. In: IL PENSIERO SCIENTIFICO (ed.), Proceedings of the Seventh International Congress of the International Society of Haematology, Rome, 1958, p. 1006.
• 8 Gros C, and Labouesse B. 1969; Study of the dansylation reaction of amino-acids, peptides and proteins. European Journal of Biochemistry 7: 463.
• 9 Hartley B. S. 1970; Strategy and tactics in Protein Chemistry. The Biochemical Journal 119: 805.
• 10 Hemker H. C, Ménaché D, and Soulier J. P. 1964 Specific Activity of Prothrombin. First Meeting of the Federation of European Biochemical Societies, Abstracts, London, p. 48.
• 11 Hirs C. H. W. 1956; The oxidation of Ribonuclease with performic acid. The Journal of Biological Chemistry 219: 611.
• 12 Howard S. M, and Pierce J. G. 1969; The Tryptic Glycopeptides of Bovine Thyrotropin: their composition and similarities to those of luteinizing hormone. The Journal of Biological Chemistry 244: 6468.
• 13 Ingwall J. S, and Sheraga H. A. 1969; Purification and Properties of Bovine Prothrombin. Biochemistry 8: 1860.
• 14 Josso F, Lavergne J. M, Weilland C, and Soulier J.P. 1967; Etude immunologique de la Prothrombine et de la Thrombine humaines. Thrombosis et Diathesis Haemorrhagica 18: 311.
• 15 Josso F, Lavergne J. M, Gouault M, Prou-Wartelle O, and Soulier S. P. 1968; Différents états moléculaires du facteur II (Prothrombine). Leur étude a l’aide de la staphy-locoagulase et d’anticorps antifacteur II. I: le facteur II chez les sujets traités par les antagonistes de la vitamine K. Thrombosis et Diathesis Haemorrhagica 20: 88.
• 16 Josso F, Monasterio de Sanchez J, Lavergne J. M, Ménaché D, and Soulier J. P. 1971; Congenital Abnormalities of the Prothrombin molecule (Factor II) in four siblings: prothrombin Barcelona. Blood 38: 9.
• 17 Lanchantin G. F, Friedmann J. A, and Hart D. W. 1967; Esterase and Clotting activities derived from citrate activation of human prothrombin. The Journal of Biological Chemistry 242: 2491.
• 18 Lanchantin G. F, Hart D. W, Friedmann J. A, Saavedra N. V, and Mehl J. W. 1968; a Amino acid composition of human plasma prothrombin. The Journal of Biological Chemistry 243: 5479.
• 19 Lanchantin G. F, Friedmann J. A, and Hart D. W. 1968; b On the occurrence of polymorphic human prothrombin. The Journal of Biological Chemistry 243: 476.
• 20 Laurell C. B. 1966; Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analytical Biochemistry 15: 45.
• 21 Lewis M. L, and Ware A. G. 1953; Simple procedure for separation of Prothrombin and accelerator globulin from citrated human plasma. Proceedings of the Society for Experimental Biology and Medicine 84: 636.
• 22 Magnusson S. 1965; a Edman degradation of components of the bovine and human prothrombin. Thrombin systems. Arkiv för Kemi 24: 375.
• 23 Magnusson S. 1965; b Purification of Prothrombin from human citrated plasma fractions II + III (Cohn’s method 6). Arkiv for Kemi 24: 367.
• 24 Magnusson S. 1970; Structure and function of Thrombin and Prothrombin. Thrombosis et Diathesis Haemorrhagica, Suppl 38: 97.
• 25 Magnusson S. 1971. Thrombin and Prothrombin. In The Enzymes. Third edn. Vol. 3. Academic Press; New York and London: 277.
• 26 Owren P. A. 1949; A quantitative one-stage method for the assay of Prothrombin. Scandinavian Journal of Clinical and Laboratory Investigation 2: 81.
• 27 Rovery M. 1972 personal communication.
• 28 Scheidegger J. J. 1955; Une microméthode de l’immunoélectrophorèse. International Archives of Allergy and applied Immunology 7: 103.
• 29 Seegers W. H, Heene D. L, and Marciniak E. 1966; a Activation of purified Prothrombin in ammonium sulfate solutions: purification of Autoprothrombin C. Thrombosis et Diathesis Haemorrhagica 15: 1.
• 30 Seegers W. H, Harmison G. R, Ivanovic N, and Heene D. 1966; b Free Boundary electrophoresis of purified Bovine Prothrombin and Thrombin. Thrombosis et Diathesis Haemorrhagica 15: 343.
• 31 Shapiro S. S, and Waugh D. F. 1966; The purification of human prothrombin. Thrombosis et Diathesis Haemorrhagica 16: 469.
• 32 Shapiro S. S, Martinez J, and Holboen R. R. 1969; Congenital Dysprothrombinemia: an inherited structural disorder of human prothrombin. The Journal of Clinical Investigation 48: 2251.
• 33 Soulier J. P, and Larrieu M. J. 1952; Etude analytique des temps de quick allongés. Dosages de prothrombine, de proconvertine et de proaccélérine. Le Sang 23: 549.
• 34 Soulier J. P, and Larrieu M. J. 1953; Nouvelle méthode de diagnostic de l’hémophilie. Dosage des facteurs antihémophiliques A et B. Le Sang 24: 205.
• 35 Soulier J. P. 1959; Un nouvel adsorbant des facteurs de coagulation: la Bentonite. Revue d’Hématologie 14: 26.
• 36 Soulier J. P, and Prou-Wartelle O. 1966; Etude comparative des taux de cofacteur de la staphylocoagulase (CRF) et des taux de facteur II (Prothrombine) dans diverses conditions. Nouvelle Revue française d’Hématologie 6: 623.
• 37 Tishkoff G. H, Williams L. C, and Brown D. M. 1968; Preparation of highly purified prothrombin complex. I Crystallization, Biological activity and molecular properties. The Journal of Biological Chemistry 243: 4151.
• 38 Tishkoff G. H, Williams L. C, and Brown D. M. 1970; Preparation and properties of human prothrombin complex. Thrombosis et Diathesis Haemorrhagica 24: 325.
• 39 Ware A. G, and Seegers W. H. 1949; Two-stage procedure for the quantitative determination of prothrombin concentration. American Journal of Clinical Pathology 19: 471.
• 40 Weber K, and Osborn M. 1969; The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. The Journal of Biological Chemistry 244: 4406.