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. 1978 Nov 1;175(2):635–641. doi: 10.1042/bj1750635

Purification and reaction mechanism of the primary inhibitor of plasmin from human plasma

Ulla Christensen 1,2, Inge Clemmensen 1,2
PMCID: PMC1186113  PMID: 154322

Abstract

The primary inhibitor of plasmin in human plasma was purified by a four-step procedure involving fractional (NH4)2SO4 precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on both a plasminogen–CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor–plasminogen complex was determined to be approx. 3μm at pH7.8. The reactions of the inhibitor with human plasmin and with bovine trypsin were studied. Comparison of the results obtained confirms the hypothesis previously presented, namely that the reaction of the inhibitor with plasmin involves at least two steps, the initial rapid formation of an enzyme–inhibitor complex followed by a slow irreversible transition to another complex. The results also indicate that the reaction of the inhibitor with trypsin involves just a single, irreversible step, so that this reaction seems to be less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (approx. 26μm) for 6-aminohexanoic acid is obtained for both its effect on the reaction of the inhibitor with trypsin and for competitive inhibition of trypsin. The inhibitory effect of 6-aminohexanoic acid thus seems to be due to its blocking of the active site of trypsin. In contrast with this, the inhibitory effects of l-lysine and 6-aminohexanoic acid on the inhibitor–plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a second site(s) on plasmin is discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chase T., Jr, Shaw E. Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters. Titration of the enzymes. Biochemistry. 1969 May;8(5):2212–2224. doi: 10.1021/bi00833a063. [DOI] [PubMed] [Google Scholar]
  2. Christensen U., Clemmensen I. Kinetic properties of the primary inhibitor of plasmin from human plasma. Biochem J. 1977 May 1;163(2):389–391. doi: 10.1042/bj1630389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Christensen U., Müllertz S. Kinetic studies of the urokinase catalysed conversion of NH2-terminal lysine plasminogen to plasmin. Biochim Biophys Acta. 1977 Jan 11;480(1):275–281. doi: 10.1016/0005-2744(77)90340-0. [DOI] [PubMed] [Google Scholar]
  4. Christensen U. pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds. Biochim Biophys Acta. 1975 Aug 26;397(2):459–467. doi: 10.1016/0005-2744(75)90136-9. [DOI] [PubMed] [Google Scholar]
  5. Collen D., De Cock, Verstraete M. Immunochemical distinction between antiplasmin and alpha-antitrypsin. Thromb Res. 1975 Jul;7(1):245–249. doi: 10.1016/0049-3848(75)90142-5. [DOI] [PubMed] [Google Scholar]
  6. Collen D. Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma. Eur J Biochem. 1976 Oct 1;69(1):209–216. doi: 10.1111/j.1432-1033.1976.tb10875.x. [DOI] [PubMed] [Google Scholar]
  7. ERLANGER B. F., KOKOWSKY N., COHEN W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys. 1961 Nov;95:271–278. doi: 10.1016/0003-9861(61)90145-x. [DOI] [PubMed] [Google Scholar]
  8. Landmann H. Studies on the mechanism of action of synthetic antifibrinolytics. A comparison with the action of derivatives of benzamidine on the fibrinolytic process. Thromb Diath Haemorrh. 1973 May 10;29(2):253–275. [PubMed] [Google Scholar]
  9. Moroi M., Aoki N. Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis. J Biol Chem. 1976 Oct 10;251(19):5956–5965. [PubMed] [Google Scholar]
  10. Müllertz S., Clemmensen I. The primary inhibitor of plasmin in human plasma. Biochem J. 1976 Dec 1;159(3):545–553. doi: 10.1042/bj1590545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Müllertz S. Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin. Biochem J. 1974 Nov;143(2):273–283. doi: 10.1042/bj1430273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Rickli E. E., Otavsky W. I. A new method of isolation and some properties of the heavy chain of human plasmin. Eur J Biochem. 1975 Nov 15;59(2):441–447. doi: 10.1111/j.1432-1033.1975.tb02472.x. [DOI] [PubMed] [Google Scholar]
  13. Wiman B., Collen D. On the kinetics of the reaction between human antiplasmin and plasmin. Eur J Biochem. 1978 Mar 15;84(2):573–578. doi: 10.1111/j.1432-1033.1978.tb12200.x. [DOI] [PubMed] [Google Scholar]
  14. Wiman B., Collen D. Purification and characterization of human antiplasmin, the fast-acting plasmin inhibitor in plasma. Eur J Biochem. 1977 Aug 15;78(1):19–26. doi: 10.1111/j.1432-1033.1977.tb11709.x. [DOI] [PubMed] [Google Scholar]
  15. Yoshida N., Sasaki A., Inoue H. An anionic trypsin-like enzyme from Streptomyces eythreus. FEBS Lett. 1971 Jun 10;15(2):129–132. doi: 10.1016/0014-5793(71)80039-x. [DOI] [PubMed] [Google Scholar]

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