Modification of a single tryptophan residue in human Cu,Zn-superoxide dismutase by peroxynitrite in the presence of bicarbonate

Citation
F. Yamakura et al., Modification of a single tryptophan residue in human Cu,Zn-superoxide dismutase by peroxynitrite in the presence of bicarbonate, BBA-PROT ST, 1548(1), 2001, pp. 38-46
Citations number
36
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
ISSN journal
01674838 → ACNP
Volume
1548
Issue
1
Year of publication
2001
Pages
38 - 46
Database
ISI
SICI code
0167-4838(20010709)1548:1<38:MOASTR>2.0.ZU;2-4
Abstract
Human recombinant Cu,Zn-SOD was reacted with peroxynitrite in a reaction mi xture containing 150 mM potassium phosphate buffer (pH 7.4) 25 mM sodium bi carbonate, 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. Am ino acid contents of the modified enzyme showed no significant difference i n all amino acids except the loss of a single tryptophan residue of the enz yme. The peroxynitrite-modified enzyme showed an increase in optical absorp tion around 350 nm and 30% reduced enzyme activity based on the copper cont ents. The modified enzyme showed the same electron paramagnetic resonance s pectrum as that of the control enzyme. The modified Cu,Zn-SOD showed a sing le protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresi s (SDS-PAGE) and five protein bands in non-denaturing PAGE. From this evide nce, we conclude that nitration and/or oxidation of the single tryptophan 3 2 and partial inactivation of the enzyme activity of Cu,Zn-SOD is caused by a peroxynitrite-carbon dioxide adduct without perturbation of the active s ite copper integrity. (C) 2001 Elsevier Science B.V. All rights reserved.