Immunological detection of N-formylkynurenine in oxidized proteins.

Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Free Radical Biology and Medicine (Impact Factor: 5.27). 06/2009; 46(9):1260-6. DOI: 10.1016/j.freeradbiomed.2009.01.020
Source: PubMed

ABSTRACT Reactions of tryptophan residues in proteins with radical and other oxidative species frequently lead to cleavage of the indole ring, modifying tryptophan residues into N-formylkynurenine (NFK) and kynurenine. Tryptophan modification has been detected in physiologically important proteins and has been associated with a number of human disease conditions. Modified residues have been identified through various combinations of proteomic analyses, tryptic digestion, HPLC, and mass spectrometry. Here we present a novel, immunological approach using polyclonal antiserum for detection of NFK. The specificity of our antiserum is confirmed using photooxidation and radical-mediated oxidation of proteins with and without tryptophan residues. The sensitivity of our antiserum is validated through detection of NFK in photooxidized myoglobin (two tryptophan residues) and in carbonate radical-oxidized human SOD1, which contains a single tryptophan residue. Analysis of photooxidized milk also shows that our antiserum can detect NFK residues in a mixture of proteins. Results from mass spectrometric analysis of photooxidized myoglobin samples corroborate the immunological data, detecting an increase in NFK content as the extent of photooxidation increases.

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