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.
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.
Yamakura
Fumiyuki
F
Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba 270-1695. yamakura@sakura.juntendo.ac.jp
Matsumoto
Takashi
T
Ikeda
Keiichi
K
Taka
Hikari
H
Fujimura
Tsutomu
T
Murayama
Kimie
K
Watanabe
Eiji
E
Tamaki
Makoto
M
Imai
Takeo
T
Takamori
Kenji
K
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*
D000596:Amino Acids; D010446:Peptide Fragments; D030421:Peroxynitrous Acid; D014364:Tryptophan; D006861:Hydrogen Peroxide; D009195:Peroxidase; D013482:Superoxide Dismutase