title: Mass spectral evidence for carbonate-anion-radical-induced posttranslational modification of tryptophan to kynurenine in human Cu, Zn superoxide dismutase.
abstract: Previously, we showed that oxidation of tryptophan-32 (Trp-32) residue was crucial for H(2)O(2)/bicarbonate (HCO(3)(-))-dependent covalent aggregation of human Cu,Zn SOD1 (hSOD1). The carbonate anion radical (CO(3)(-))-induced oxidation of Trp-32 to kynurenine-type oxidation products was proposed to cause the aggregation of hSOD1. Here we used the matrix-assisted laser desorption ionization-time of flight mass spectroscopy, high-performance liquid chromatography-electrospray ionization mass spectroscopy, and liquid chromatography mass spectroscopy methods to characterize products. Results show that a peptide region (31-36) of hSOD1 containing the Trp-32 residue (VWGSIK) is oxidatively modified to the N-formylkynurenine (NFK)- and kynurenine (Kyn)-containing peptides (V(NFK)GSIK) and (V(Kyn)GSIK) during HCO(-)-dependent peroxidase activity of hSOD1. Also, UV photolysis of a cobalt complex that generates authentic CO(3)(-) radical induced a similar product profile from hSOD1. Similar products were obtained using a synthetic peptide with the same amino acid sequence (i.e., VWGSIK). We propose a mechanism involving a tryptophanyl radical for CO(3)(-)-induced oxidation of Trp-32 residue (VWGSIK) in hSOD1 to V(NFK)GSIK and V(Kyn)GSIK.
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Joy
authors.initials: J
authors.lastname: Joseph
authors.forename: John
authors.initials: J
authors.lastname: Crow
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000838:Anions / Q000737:chemistry / Q000494:pharmacology; D002254:Carbonates / Q000737:chemistry / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007737:Kynurenine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
chemical_list: D000838:Anions; D002254:Carbonates; D005609:Free Radicals; D007737:Kynurenine; D003300:Copper; D014364:Tryptophan; D013482:Superoxide Dismutase; D015032:Zinc