title: Identification of site(s) of insulin nitration by peroxynitrite and characterization of its structural change.
abstract: Insulin nitration mediated by peroxynitrite (ONOO(-)) has been implicated in diabetes and diabetic cardiovascular complications. In this study, we identified the nitration sites of porcine insulin by infusion of ONOO(-) and quantified its secondary structural change. Insulin was cleaved with V8 protease to six peptides (four of them contained each tyrosine residue), then analyzed by HPLC-MS and further confirmed the nitration sites by HPLC-MS/MS. At low accumulated doses of peroxynitrite, the main products were two different mono-nitrated insulin species at Tyr-A19 and Tyr-B26 with Tyr-A19 being predominant as shown by peptide mapping. Also, the content of alpha-helix structure of insulin reduced to 22.9 % and random-coil structure increased to 30.2 % (compare with native insulin of 41.7 % and 13.7 %, respectively) as determined by FTIR spectra.
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100022, P R China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Luo
authors.forename: R
authors.initials: R
authors.lastname: Zhong
authors.forename: D
authors.initials: D
authors.lastname: Gao
authors.forename: S
authors.initials: S
authors.lastname: Cui
mesh_terms: D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism* / Q000494:pharmacology; D017433:Protein Structure, Secondary / Q000187:drug effects; D020539:Sequence Analysis, Protein; D017550:Spectroscopy, Fourier Transform Infrared; D053719:Tandem Mass Spectrometry
chemical_list: D007328:Insulin; D030421:Peroxynitrous Acid