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The use of t-butyl hydroperoxide as a probe for methionine oxidation in proteins.

Abstract

The susceptibility of native recombinant interferon gamma (rIFN-gamma, Actimmune) and recombinant tissue-type plasminogen activator (rt-PA, Activase) to methionine oxidation when treated with the oxidizing agent t-butyl hydroperoxide (TBHP) was investigated. The results showed that two of the five methionine residues in rIFN-gamma were susceptible to oxidation by TBHP, while three of the five methionines in rt-PA were found to be oxidizable. The oxidized methionine residues were found to be in the sulfoxide [Met(O)] form, and no other residue(s) appeared to be modified during the TBHP treatment. These results also showed that during treatment of a native protein with TBHP only the exposed methionine residues were oxidized. The biological activity of both molecules were unaffected by the treatment with TBHP. A comparative study between TBHP and hydrogen peroxide (H2O2) demonstrated that H2O2 was also a methionine-specific oxidizer. However, this study also showed that H2O2 was not able to distinguish between exposed and buried methionine residues, as significant portions of all five methionine residues in native rIFN-gamma were oxidized by treatment with H2O2. TBHP should be useful for identifying surface methionine residues in a protein of unknown structure and a valuable reagent for methionine oxidation in pharmaceutical stability studies.

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  • Publisher Full Text
  • Publisher Full Text
  • Authors

    Keck RG

    Source

    Analytical biochemistry 236:1 1996 Apr 5 pg 56-62

    MeSH

    Amino Acid Sequence
    Biological Assay
    Fibrinolysis
    HLA-DR Antigens
    Humans
    Hydrogen Peroxide
    Interferon-gamma
    Mass Spectrometry
    Methionine
    Molecular Sequence Data
    Oxidation-Reduction
    Peptide Fragments
    Peroxides
    Proteins
    Recombinant Proteins
    Tissue Plasminogen Activator
    tert-Butylhydroperoxide

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    8619496