Download PDF
Thromb Haemost 1960; 4(02): 167-177
DOI: 10.1055/s-0038-1654494
Originalarbeiten — Original Articles — Travaux Originaux
Schattauer GmbH

N-terminal Glycine Analysis for the Determination of the Proteolytic Activity of Thrombin[*]

Staffan Magnusson (With the Technical Assistance of Miss Elisabeth Borelius)
1   Chemistry Department II, Karolinska Institutet, Stockholm, Sweden
› Author Affiliations
Further Information

Publication History

Publication Date:
17 June 2018 (online)

Also available at
    Permissions and Reprints

Summary

In the method presented in this paper for the assay of thrombin activity, N-terminal amino acids are determined with the phenylisothiocyanate method of Edman. Highly purified bovine fibrinogen is proteolyzed by thrombin under strictly defined conditions. The increase in N-terminal glycine is then directly proportional to the activity of the thrombin. Five different thrombin preparations were standardized with this N-terminal method and with a clotting method. The results obtained with the two methods are in good agreement. Because the N-terminal method is accurate and highly specific for thrombin it is proposed as standard method for the chemical determination of thrombin activity. One NIH unit of thrombin was found to make 0.4 μimoles of glycine appear in N-terminal position.

* Supported by grants from the Swedish National Association against Tuberculosis and other Social Diseases and from the Swedish Society for Medical Research.


 

  • References

  • (1) Bailey K, Bettelheim F. R, Lorand L, Middlebrook W. R. Action of thrombin in the clotting of fibrinogen. Nature 167: 233 1951;
    CrossrefPubMedGoogle Scholar
  • (2) Bettelheim F. R, Bailey K. The products of the action of thrombin on fibrinogen. Biochim. biophys. Acta 9: 578 1952;
    CrossrefPubMedGoogle Scholar
  • (3) Blombäck B, Yamashina I. On the N-terminal amino acids in fibrinogen and fibrin. Ark. Kemi 12: 299 1958;
    PubMedGoogle Scholar
  • (4) Blombäck B. Studies on the action of thrombic enzymes on bovine fibrinogen as measured by N-terminal analysis. Ark. Kemi 12: 321 1958;
    PubMedGoogle Scholar
  • (5) Jorpes E, Vrethammar T, Öhman B, Blombäck B. On the assay of thrombin preparations. J. Pharm. (London) 10: 561 1958;
    CrossrefPubMedGoogle Scholar
  • (6) Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Ark. Kemi 10: 415 1956;
    PubMedGoogle Scholar
  • (7) Edman P. Preparation of phenylthiohydantoins from some natural amino acids. Acta chem. scand. 4: 277 1950;
    CrossrefPubMedGoogle Scholar
  • (8) Sjöquist J. Paper strip identification of phenylthiohydantoins. Acta chem. scand. 7: 447 1953;
    CrossrefPubMedGoogle Scholar
  • (9) Edman P, Sjöquist J. Identification and semiquantitative determination of phenyl thiohydantoins. Acta chem. scand. 10: 1507 1956;
    CrossrefPubMedGoogle Scholar
  • (10) Sjöquist J. Determination of amino acids as phenylthiohydantoin derivatives. III. Quantitative determination of 3-phenyl-2-thiohydantoins from paper chromatograms. Biochim. biophys. Acta. accepted for publication
    PubMedGoogle Scholar
  • (11) Sjöquist J. Determination of amino acids as phenylthiohydantoin derivatives. I. Micro synthesis of 3-phenyl-2-thiohydantoins from amino acids. Ark. Kemi 11: 129 1957;
    PubMedGoogle Scholar
  • (12) Sherry S, Troll W. The action of thrombin on synthetic substrates. J. biol. Chem. 208: 95 1954;
    PubMedGoogle Scholar
  • (13) Martin C. J, Golubow J, Axelrod A. E. The hydrolysis of carbo-benzoxy-L-tyrosine p-nitrophenyl ester by various enzymes. J. biol. Chem. 234: 1718 1959;
    PubMedGoogle Scholar
  • (14) Miller K. D. Reversible dénaturation of thrombin activities in urea solution. Ann. Report, Div. Labs. Res., N. Y.. State Dept. Health; Albany: 44 1957
    Google Scholar
  • (15) Wallen P, Bergström K. Action of thrombin on plasmin digested fibrinogen. Acta chem. scand. 12: 574 1958;
    CrossrefPubMedGoogle Scholar