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.
Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. ehrensh1@niehs.nih.gov
Marilyn
M
Ehrenshaft
Sueli Oliveira
SO
Silva
Irina
I
Perdivara
Piotr
P
Bilski
Robert H
RH
Sik
Colin F
CF
Chignell
Kenneth B
KB
Tomer
Ronald P
RP
Mason
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