Identification of the nitration site of insulin by peroxynitrite.

Our previous investigation indicated that insulin can be nitrated by peroxynitrite in vitro. In this study, the preferential nitration site of the four tyrosine residues in insulin molecule was confirmed. Mononitrated and dinitrated insulins were purified by RP-HPLC. 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.