Characterization of Human Glycated DNA modified with Peroxynitrite
Keywords:Glycated DNA, Peroxynitrite, Human DNA
Glycation is the non-enzymatic addition or insertion of sugar molecules into DNA, proteins and lipids that occur in biological molecules. The glycation of DNA gives rise to characteristic nucleotide adducts, some of which have been found to increase in oxidative stress. The glycation causes damage to DNA which is associated with mutagenesis, carcinogenesis and is also considered to be a pathogenic factor for diabetes mellitus. The present study was designed to evaluate the influence of glucose on DNA isolated from human blood and its modification with peroxynitrite. Modified, glycated DNA was characterized by UV spectroscopy, melting temperature and electrophoretic profile. Oxidation incurred by glycation of DNA and peroxynitrite was also analyzed by scavenging studies. In the present study peroxynitrite of glycated DNA leads to damage which may be due to nitration, deamination along with the formation of strand break and mutation. The modified DNA showed 18% hyperchromicity effect with the peak shift of approximately 5nm to higher wavelength, which may be due to alteration in normal helical structure, and exposure of bases causes marked absorption in the UV range. Agarose gel electrophoresis clearly demonstrates that the instability in ?-helix is because of peroxynitrite modification of glycated DNA. Additional evidence for the structural perturbation in DNA were further analyzed by thermal denaturation study. Peroxynitrite modification showed significant decrease in Tm which could be attributed to single strand generation.