Identification of site(s) of insulin nitration by peroxynitrite and characterization of its structural change.
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.
Wang
Y
Y
College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100022, P R China.
Luo
Y
Y
Zhong
R
R
Gao
D
D
Cui
S
S
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
D007328:Insulin; D030421:Peroxynitrous Acid