Effects Of Nitration On The Structure And Function Of Insulin

Diabetes is a common kind of metabolic diseases. There is emerging evidence that RNS/ROS make a significant contribution to the progression of diabetes and its complications. Protein tyrosine nitration is an important posttranslational modification, and involved in a variety of diseases. Nitration by peroxynitrite is a principal pathway, and nitrotyrosine is formed by free radical reaction. Diabetes-associated protein nitration has been reported by several recent publications. Insulin is one of the most important versatile hormones, and it may be a potential target of peroxynitrite during conditions of oxidative stress in pancreatic isletβ-cells.In this paper the nitration of insulin by peroxynitrite in vitro and the effects of nitration on the structure and function of insulin were investigated. The influence of antioxidants and some facts on insulin nitration were also studied. The main results obtained were as follows:(1) The products of insulin nitration by peroxynitrite were characterized by UV-Vis, Native-PAGE, Western blotting and MALDI-TOF-MS, and the influence of the pH value, carbon dioxide, iron complexes and albumin was also investigated. The results showed that insulin could be nitrated by peroxynitrite in vitro, and with increasing the concentration of peroxynitrite all of the four tyrosine residues in insulin could be nitrated. The nitration reaction was correlated with the pH value of the solution, and at physiological pH peroxynitrite has the maximum nitration capability. Carbon dioxide and Fe(III)-EDTA could catalyze the nitration reaction, but Fe(III)-DTPA had no effect. Albumin could competitively inhibit the nitration of insulin by peroxynitrite, but in the presence of high concentrations of albumin insulin could still be nitrated.(2) Mononitrated and dinitrated insulin were purified by RP-HPLC. The preferential nitration site of the four tyrosine residues in insulin was confirmed by Native-PAGE, RP-HPLC and MALDI-TOF-MS. Following reduction of insulin disulfide bridges, Native-PAGE indicated that A-chain was preferentially nitrated. Combination of enzymatic digestion of mononitrated insulin with endoproteinase Glu-C, mass spectrometry confirmed that Tyr-A14 was the preferential nitration site when insulin was treated with peroxynitrite. Tyr-A19 maybe was the next preferential nitration site. According to the crystal structure, Tyr-B26 between the two tyrosine residues in insulin B-chain was likely easier to be nitrated by peroxynitrite.(3) The effects of nitration on the secondary structures of insulin were investigated by analysis of the fluorescence spectra, circular dichroism spectra and fourier transformation infrared spectra of the nitrated insulin. The contents ofα-helix of the mono and dinitrated insulin were decreased, and the nitration of A-chain could make the 310 helix of A(12-17) segment convert to irregular helix or unordered structures, but it didn't influence the main part of the insulin binding surface with insulin receptor, and the nitration of Tyr-A19 may alter the hydrophobicity of the insulin binding surface. In the mean time the introduction of nitro group could incline the mono and dinitrated insulin to aggregation.The voltammetric response of Tyr and NT was investigated by cyclic voltammetry, and the results showed that introduction of nitro group could alter the hydrogen bonding manner of the phenol hydroxyl and influence the interaction between Tyr and albumin. These reflected that nitration could affect the binding between insulin and insulin receptor to some extent.(4) The biological activity of mononitrated insulin was assayed, and the phosphorylation of insulin receptor by stimulation of nitrated insulin was also investigated. The receptor binding capability of mononitrated insulin was about 70% of that of insulin, and the biological activity in vivo accorded with it.(5) The effects of antioxidants on the nitration of insulin by peroxynitrite were evaluated by determination of the production of nitrotyrosine by UV-Vis, and the interaction of GSH and Ebselen and the effects on the nitration of insulin were investigated by RP-HPLC and ESI-MS. Oleic acid and arachidonic acid have strong inhibition effects on nitration of insulin, and the inhibition capability of unsaturated fatty acids were increasing with the unsaturation degree. At the experimental concentration range sodium selenite had no effect on nitration of insulin. Individual GSH, VC, Ebselen and VE had different inhibition capabilities on nitration of insulin, but at a definite concentration range antagonism was occurred between GSH and Ebselen because an adduct, Ebselen-Se-SG, could be formed.