The Nitration Of Insulin By Peroxynitrite In Vitro And The Influence Of Some Factors On The Nitration

Peroxynitrite is a powerful oxidizing and nitrating agent. It can cause cellular and tissue injury by multiple pathways. The nitration of tyrosine or protein tyrosine residues generates 3-nitrotyrosine is one of major pathways that peroxynitrite damages biological system. At present, it has been found that 3-nitrotyrosine is involved in more than 50 diseases, such as shock, atherosclerosis, rheumatic arthritis, amyotrophic lateral sclerosis and so on. Insulin is an important multi-function protein hormone in vivo and may be an action target of peroxynitrite, so it has an important academic significance to study on the nitration of insulin by peroxynitrite and the influence of some factors on the nitration, which could provide some evidences to treat with insulin dependent diabetes. In this paper, we mostly study on the nitration of insulin by peroxynitrite and the effects of several factors on the insulin nitration. The main results are as follows: 1)UV-Vis, MALDI-TOF-MS and native PAGE demonstrated insulin could be nitrated by peroxynitrite in vitro and the nitration was intimately related with pH. Maximum yield of 3-nitrotyrosine was obtained at physiological pH, the yield of 3-nitrotyrosine decreased significantly in more acid or basic condition. Carbon dioxide could catalyze the reaction of insulin with peroxynitrite markedly, but serum albumin and glucose could inhibit the nitration. 2)The influence of some antioxidants including GSH, ebselen, ascorbic acid, α-tocopherol and Na₂SeO₃ on insulin nitrtation by peroxynitrite were investigated. The results showed that GSH and ascorbic acid had a stronger inhibition than ebselen and α-tocopherol on the nitration. However Na2SeO3 had no obvious effect on the reaction in the same conditions. Although GSH or ebselen could inhibit the nitration respectively, there was an antagonism between them, because an adduct, ebselen-Se-SG, is formed. Unsaturated fatty acid including oleic acid and arachidonic acid could also inhibit insulin nitration by peroxynitrite significantly. 3)The effect of iron complexes on the nitration by peroxynitrite was studied. At physiological pH, Fe3+EDTA could catalyze the reaction of insulin nitration by peroxynitrite, and the ferric citrate took second place, but Fe3+DTPA had no significant effect on the nitration. CO2 could suppress the catalysis of Fe3+EDTA on the nitration of insulin. The mechanism of iron complexes promoting insulin nitration may be by electrophilic aromatic nitration.