© 2005 The Japanese Biochemical Society
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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
1 Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba 270-1695; 2 Department of Food Science and Nutrition, Showa Women’s University, Setagaya-ku, Tokyo 154-8533; 3 The Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, and 7 Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Chiba 279-0021; 4 Division of Proteome and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo 113-8421; 5 Department of Chemistry, Faculty of Science, Toho University, Miyama, Funabashi, Chiba 274-8510; and 6 Department of Life Science and Graduate School of Life Science, Rikkyo (St. Paul’s) University, Toshima-ku, Tokyo 171-8501
* To whom correspondence should be addressed. Tel: +81-476-98-1001, Fax: +81-476-98-1011, E-mail: yamakura{at}sakura.juntendo.ac.jp
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.
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