The Use of t-Butyl Hydroperoxide as a Probe for Methionine Oxidation in Proteins

Publisher： Elsevier

ISSN： 0003-2697

Source： Analytical Biochemistry, Vol.236, Iss.1, 1996-04, pp. : 56-62

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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 (H 2 O 2 ) demonstrated that H 2 O 2 was also a methionine-specific oxidizer. However, this study also showed that H 2 O 2 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 H 2 O 2 . 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.