Modification of a single tryptophan residue in human Cu,Zn-superoxide dismutase by peroxynitrite in the presence of bicarbonate.
Human recombinant Cu,Zn-SOD was reacted with peroxynitrite in a reaction mixture containing 150 mM potassium phosphate buffer (pH 7.4) 25 mM sodium bicarbonate, and 0.1 mM diethylenetriamine pentaacetic acid. Disappearance of fluorescence emission at 350 nm, which could be attributed to modification of a single tryptophan residue, was observed in the modified enzyme with a pH optimum of around 8.4. A fluorescence decrease with the same pH optimum was also observed without sodium bicarbonate, but with less efficiency. Amino acid contents of the modified enzyme showed no significant difference in all amino acids except the loss of a single tryptophan residue of the enzyme. The peroxynitrite-modified enzyme showed an increase in optical absorption around 350 nm and 30% reduced enzyme activity based on the copper contents. The modified enzyme showed the same electron paramagnetic resonance spectrum as that of the control enzyme. The modified Cu,Zn-SOD showed a single protein band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and five protein bands in non-denaturing PAGE. From this evidence, we conclude that nitration and/or oxidation of the single tryptophan 32 and partial inactivation of the enzyme activity of Cu,Zn-SOD is caused by a peroxynitrite-carbon dioxide adduct without perturbation of the active site copper integrity.
Yamakura
F
F
Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba, Japan. yamakura@sakura.juntendo.ac.jp
Matsumoto
T
T
Fujimura
T
T
Taka
H
H
Murayama
K
K
Imai
T
T
Uchida
K
K
D000596:Amino Acids / Q000032:analysis; D001639:Bicarbonates / Q000737:chemistry*; D002245:Carbon Dioxide / Q000737:chemistry; D003300:Copper / Q000032:analysis; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008958:Models, Molecular; D009566:Nitrates / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D001639:Bicarbonates; D009566:Nitrates; D011994:Recombinant Proteins; D002245:Carbon Dioxide; C039642:peroxynitric acid; D003300:Copper; D014364:Tryptophan; D013482:Superoxide Dismutase; D015032:Zinc