title: Nitrated and oxidized products of a single tryptophan residue in human Cu,Zn-superoxide dismutase treated with either peroxynitrite-carbon dioxide or myeloperoxidase-hydrogen peroxide-nitrite.
abstract: We reported previously that a single tryptophan residue, Trp32, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, 38-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp32 was the only significant amino acid lost. After trypsin digestion of the modified SOD with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), 32, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-3-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about 30% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.
authors.affiliation: Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba 270-1695. yamakura@sakura.juntendo.ac.jp
authors.forename: Fumiyuki
authors.initials: F
authors.lastname: Yamakura
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ikeda
authors.forename: Hikari
authors.initials: H
authors.lastname: Taka
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Fujimura
authors.forename: Kimie
authors.initials: K
authors.lastname: Murayama
authors.forename: Eiji
authors.initials: E
authors.lastname: Watanabe
authors.forename: Makoto
authors.initials: M
authors.lastname: Tamaki
authors.forename: Takeo
authors.initials: T
authors.lastname: Imai
authors.forename: Kenji
authors.initials: K
authors.lastname: Takamori
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D030421:Peroxynitrous Acid; D014364:Tryptophan; D006861:Hydrogen Peroxide; D009195:Peroxidase; D013482:Superoxide Dismutase