Immunological detection of N-formylkynurenine in oxidized proteins

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 radicalmediated 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 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.

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions

“…It is also plausible that the initial reaction involves the reaction of L-Trp with an IDO protein radical (77). Upon formation, the neutral tryptophanyl radical is known to react with O 2 to form a peroxyl radical (75,77,78), which upon forming a hydroperoxide can rearrange via yet to be characterized chemistry into N-formylkynurenine that hydrolyzes into kynurenine (79,80). Further evidence for compound I-mediated oxidation of L-Trp is the observation that tempol and ascorbate both inhibited H 2 O 2 -induced oxidation of the amino acid by IDO (Fig.…”

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization

“…MiniSOG was also recently demonstrated to facilitate the measurement of distances of proteins within complexes in live cells by singlet oxygen triplet energy transfer of a fluorescent singlet oxygen detector on two putative interacting proteins (35). Furthermore, ligand-proximal photooxidation techniques may be modifiable with alternatives to BH such as antibody pull-down of proteins containing methionine sulfoxide or N-formylkynurenine (36,37). Future studies will address if molecules with higher affinity toward human laminin subunits α4, α5, or α2 can generate greater clinically useful nerve contrast.…”

Cardiovascular Redox and Ox Stress Proteomics

“…Using 500 lM to 1 mM H 2 O 2 , hydroxytryptophan modifications can be induced in cytochrome C oxidase, subunit 1 (158). Other oxidation end-products of Trp include NFK and KYN, which are formed after a direct interaction with 1 O 2 , or ozone (70). Two other radical reactions by which NFK and KYN can be formed are (i) the Trp radical interacting with superoxide to form Trp hydroperoxide and (ii) the Trp radical interacting with molecular oxygen, leading to a Trp peroxyl radical.…”

“…Two other radical reactions by which NFK and KYN can be formed are (i) the Trp radical interacting with superoxide to form Trp hydroperoxide and (ii) the Trp radical interacting with molecular oxygen, leading to a Trp peroxyl radical. The trytophan hydroperoxide and Trp peroxyl radical can be rearranged to form NFK and KYN (70). Similar to some amino acids such as Met, Trp may also theoretically act as a longrange electron transfer shuttle between other amino acids such as Tyr and Cys, which would allow for the formation of tyrosyl and thiyl radicals, respectively, on the peptide backbone (256).…”

“…Similar to some amino acids such as Met, Trp may also theoretically act as a longrange electron transfer shuttle between other amino acids such as Tyr and Cys, which would allow for the formation of tyrosyl and thiyl radicals, respectively, on the peptide backbone (256). Antibodies have been designed for the detection of NFK on protein side chains (70,267), but MS detection suggests that oxidized Trp products can sometimes be a result of artifacts, as they have been observed with proteins isolated by gel electrophoresis, but not with in-solution digests (214).…”