Peroxynitrite reaction with eye lens proteins: alpha-crystallin retains its activity despite modification.
OBJECTIVE
. Peroxynitrite is a highly potent reactive oxygen/nitrogen species present in the environment and also endogenously in the eye, that causes a variety of disorders. This study was undertaken to look at the oxidative damage that peroxynitrite causes to the proteins of the lens and the functional consequences thereof.


METHODS
. Peroxynitrite was allowed to react with alpha-, beta-, and gamma-crystallins. The formation of nitrotyrosine and nitrotryptophan, dityrosine, protein covalent cross-links, and chain degradation products were monitored by spectroscopy and SDS-PAGE. Conformational changes occurring in the protein were monitored with circular dichroism spectroscopy. The chaperoning ability of alpha-crystallin was assayed by monitoring its ability to inhibit the self-aggregation of two test proteins: beta-crystallin and insulin.


RESULTS
. Peroxynitrite reaction produced nitrotyrosine, nitrotryptophan, and dityrosine, nondisulfide covalent cross-linked aggregates, and peptide chain degradation. The hydroxyl radicals produced by peroxynitrite caused more chain degradation than did the carbonate radicals. The oxidative reaction caused increased conformational disorder. The yield was highest in gamma-crystallin and least in alpha-crystallin. The chaperoning ability of alpha-crystallin was not affected.


CONCLUSIONS
. Peroxynitrite reacts with lens proteins, causing extensive covalent chemical changes. However, alpha-crystallin retains its chaperoning ability, despite the oxidative changes caused by the peroxynitrite reaction, indicating its functional robustness.
Thiagarajan
Geetha
G
Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India.
Lakshmanan
Jaganathan
J
Chalasani
Madhavilatha
M
Balasubramanian
Dorairajan
D
D000818:Animals; D002417:Cattle; D007908:Lens, Crystalline / Q000737:chemistry*; D018832:Molecular Chaperones / Q000737:chemistry; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000737:chemistry*; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis; D038201:alpha-Crystallins / Q000737:chemistry* / Q000528:radiation effects
D018832:Molecular Chaperones; D038201:alpha-Crystallins; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D014364:Tryptophan; C007543:dityrosine