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Related Experiment Videos

Evidence for an ester bond between thrombin and heparin cofactor.

W G Owen, G D Penick, E Yoder

    Thrombosis and Haemostasis
    |February 29, 1976
    PubMed
    Summary

    This study reveals that the thrombin inhibitor, heparin cofactor, binds to thrombin via a covalent bond. This bond is identified as a carboxylic ester, dissociable by hydroxylamine or alkaline conditions.

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    Area of Science:

    • Biochemistry
    • Proteomics
    • Biotechnology

    Background:

    • Heparin cofactor is a crucial thrombin inhibitor found in human plasma.
    • Understanding the binding mechanism between heparin cofactor and thrombin is vital for anticoagulation research.
    • Previous studies suggested a covalent bond formation in the complex.

    Purpose of the Study:

    • To elucidate the nature of the binding between heparin cofactor and thrombin.
    • To identify the type of bond involved in the heparin cofactor-thrombin complex formation.
    • To investigate the stability and dissociation conditions of the complex.

    Main Methods:

    • Purification of heparin cofactor from human plasma using affinity chromatography on heparin-agarose.
    • Treatment of the heparin cofactor-thrombin complex with guanidinium chloride, hydroxylamine, and dilute alkali.

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  • Analysis of complex dissociation using gel chromatography and dodecyl sulfate denaturation.

    • Main Results:

    • The heparin cofactor-thrombin complex remained intact in 6 M guanidinium chloride, supporting covalent bond formation.
    • Treatment with hydroxylamine dissociated the dodecyl sulfate-denatured complex into free thrombin and heparin cofactor.
    • The complex was also dissociated in dilute sodium hydroxide (pH 12).

    Conclusions:

    • The binding between heparin cofactor and thrombin involves a covalent bond.
    • The covalent bond is identified as a carboxylic ester.
    • The carboxylic ester bond can be cleaved by hydroxylamine under denaturing conditions or by alkaline hydrolysis.