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Modification of papain with tetranitromethane

S Tsukamoto, M Ohno

    Journal of Biochemistry
    |December 1, 1978
    PubMed
    Summary

    Tetranitromethane (TNM) treatment of papain causes polymerization via tyrosine cross-links, retaining esterolytic activity. This study identifies nitrated tyrosine residues and clarifies inactivation mechanisms, ruling out tryptophan involvement.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Protein Chemistry

    Background:

    • Papain, a cysteine protease, is known to polymerize.
    • Previous studies suggested tryptophan residues might be involved in papain inactivation.

    Purpose of the Study:

    • To investigate the polymerization of papain induced by tetranitromethane (TNM).
    • To identify the specific tyrosine residues involved in cross-linking.
    • To elucidate the mechanism of papain inactivation by TNM.

    Main Methods:

    • Papain modification with TNM at optimal pH (9.0).
    • Circular dichroic spectroscopy to analyze structural changes.
    • Esterolytic activity assays using benzoyl-L-arginine ethyl ester (BAEE).
    • Spectrophotometric and amino acid analyses to identify nitrated residues.

    Main Results:

    • Papain polymerization occurred through intermolecular covalent linkages involving tyrosine residues.
    • Polymerized papain retained significant esterolytic activity, with unchanged Km up to hexamer formation.
    • Tyrosine residues at positions 61, 116, 103, and 4 were identified as nitrated.
    • Oxidation of an essential sulfhydryl group occurred, leading to irreversible inactivation at higher TNM concentrations.

    Conclusions:

    • TNM-induced papain polymerization involves specific tyrosine residues.
    • Papain's enzymatic activity is largely preserved after polymerization.
    • Inactivation is linked to sulfhydryl oxidation, not tryptophan nitration.

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