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.

Links

Publisher Full Text

Publisher Full Text

Authors

Keck RG

Institution

Department of Analytical Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California, 94080-4990, USA.

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, Recombinant
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