Constructing Evaluation System For Protein Nitration And Inhibition

Peroxynitrite is one kind of important active oxygen species. Its proposed actions comprise lipid peroxidation, DNA damage, and protein modifications in cells. Protein nitration is an important pathway. As the marking substance of peroxynitrite injury in vivo, 3-nitrotyrosine has normally been detected in a variety of diseases. Therefore, to construct evaluation system for protein nitration and inhibition is very significant.Using BSA(bovine serum albumin) as nitrated substrate, peroxynitrite as nitrification reagent and melatonin as inhibitor, the evaluation system for protein nitration and inhibition was established. Subsequently, the system was verified by rutin and proanthocyanidins as inhibitors. Finally, SD diabetic rat was selected as the animal model to study the influence of melatonin on diabetes, especially the protective effect on renal.It was observed that the optimum conditions for complete nitration were temperature 37℃, pH7.2, 90min for reaction time, peroxynitrite concentration of 1.8×10^-3mol/L and the concentration ratio of bovine serum albumin and peroxynitrite was 1:18. Under these conditions, it is displayed that addition of melatonin before the process can efficiently retard the reaction. Three natural antioxidants can effectively inhibit protein nitration. The order of inhibiting ability was: melatonin > proanthocyanidins > rutin. The reduction of maximum nitration was 80.7%, 66.9% and 38.7% respectively. Finally, it is verified that mechanism of inhibiting nitration reaction was to clear away free radicals rather than to restore the nitration product.The results of animal experiments have shown that, the kidneys had been injuries in model rats of diabetes mellitus. After melatonin treatment, the organ coefficient decreased and the diabetes symptoms alleviated. Compared with the model group, the content of NO₂^-+NO₃^- and MDA in interventional group rats'tissue and serum decreased significantly. These findings suggest that melatonin can inhibit the oxidative stress response and holds substantial remission for the treatment of DN.