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Spectrophotometric assay for the quantitation of methionine sulfoxide in proteins
Author(s) -
Zaidan George S.,
Levine Rodney L.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb61-b
Subject(s) - methionine sulfoxide , methionine , sulfoxide , chemistry , cysteine , methionine sulfoxide reductase , biochemistry , amino acid , msra , cystine , iodide , organic chemistry , enzyme
Cysteine and methionine are the two sulfur‐containing amino acids normally found in proteins. Both amino acids are readily and reversibly oxidized, forming cystine and methionine sulfoxide respectively. Reversible oxidation and reduction of cysteine is implicated in catalysis, protein structure, redox sensing and regulation, and antioxidant defenses. In contrast, the functional roles of reversible oxidation of methionine remain speculative. However, the existence of methionine sulfoxide reductases in almost all organisms, from microbes through mammals suggests that these roles are important. In particular, redox cycling of methionine residues in proteins would constitute an antioxidant defense system capable of scavenging reactive oxygen species. Experimental investigation of the function of methionine sulfoxide is hampered by the lack of assays for methionine sulfoxide in intact proteins. Attempts by various laboratories to raise antibodies to methionine sulfoxide residues have been unsuccessful. Thus, we undertook the development of a chemical assay for methionine sulfoxide in proteins. Methionine sulfoxide is an oxidizing agent, capable of converting iodide to iodine:Methionine Sulfoxide + 2 I −+ 2 H +→Methionine + I 2 + H 2 OTo assay methionine sulfoxide, the protein and potassium iodide solutions are mixed, dried in a vacuum centrifuge, and exposed to trifluroroacetic acid vapor for 2–10 minutes. Addition of polyvinyl alcohol leads to formation of a red iodine complex, with an absorbance peak at 485 nm and a molar absorbtivity of 41,900 M −1 cm −1 . The method was validated with two proteins of varying but known methionine sulfoxide content, aprotinin (6.5 kD) and glutamine synthetase (52 kD). We are now working to adapt the method for the detection and quantitation of methionine sulfoxide in gels and blots.

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