Immunological detection of N-formylkynurenine in oxidized proteins.
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.
Ehrenshaft
Marilyn
M
Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. ehrensh1@niehs.nih.gov
Silva
Sueli Oliveira
SO
Perdivara
Irina
I
Bilski
Piotr
P
Sik
Robert H
RH
Chignell
Colin F
CF
Tomer
Kenneth B
KB
Mason
Ronald P
RP
D000818:Animals; D000906:Antibodies / Q000276:immunology; D006241:Haptens / Q000737:chemistry; D006801:Humans; D007106:Immune Sera; D007114:Immunization; D007118:Immunoassay; D007737:Kynurenine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D008027:Light; D013058:Mass Spectrometry; D008892:Milk / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis; D010047:Ovalbumin / Q000737:chemistry; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D012680:Sensitivity and Specificity; D013482:Superoxide Dismutase / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
D000906:Antibodies; D006241:Haptens; D007106:Immune Sera; D009211:Myoglobin; D011506:Proteins; C000606290:SOD1 protein, human; C007772:N'-formylkynurenine; D007737:Kynurenine; D014364:Tryptophan; D010047:Ovalbumin; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1