Peroxynitrite (ONOO(-)), formed by the reaction between
nitric oxide (NO) and
superoxide (O2(-)), has been implicated in the etiology of numerous disease processes.
Peroxynitrite interacts with
DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on
DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with
peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid
DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of
antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA.
Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when
peroxynitrite reacted with
DNA was
8-nitroguanine, a specific marker for
peroxynitrite induced DNA damage in inflamed tissues. The concentration of
8-nitroguanine was found to be 3.8 μM. Sera from
diabetes type 1 patients from different age groups were studied for their binding to native and
peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of
peroxynitrite modified plasmid, as compared to the native form, by auto-
antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes
autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-
peroxynitrite-modified plasmid
IgG was used as a probe to detect nitrosative lesions in the
DNA isolated from diabetes patients.