Sites Identification, Structure And Activity Analysis Of Nitrated Injury Of Fibrinogen

As an important protein in plasma involving in clotting, fibrinogen is sensitive tonitrative injury induced by a cytotoxic material-peroxynitrite(ONOO-) generated invivo, thus changing the structure and function of protein. The modification ofperoxynitrite on fibrinogen resulted in dysfunction of blood coagulation, which isassociated with related diseases. The nitration level of human fibrinogen is normallylower, but in some pathological conditions, such as atherosclerosis, myocardialinfarction, coronary heart disease, the nitration level rises rapidly. Therefore, it isextremely vital to study the nitrification mechanism and analyze resulting activity forprevention and control of cardiovascular disease.This paper used fibrinogen as a target to investigate the change of biologicalactivity triggered by ONOO--induced structure injury and modification. We utilizedseveral methods, for example, UV-Vis, fluorimetry, HPLC, LC-MS/MS and infraredspectrometer and simulated the microenvironment in vivo. The study initially clarifiedthe relevant mechanism of nitration injury and chemical modification, even provide acertain theoretical basis for the drug research. In addition, different amino acids andradicals have their special chemical modifiers, the research of the relation betweenthis groups and activity is aimed at demonstrating the effect of chemical modificationon the function and nitration of fibrinogen.The secondary structure of nitrated protein was qualitatively andquantitatively estimated by ATR-FTIR. Results indicated that for the helix and β-sheetstructure, there was an obvious shift in the amide I band in FTIR spectra, moreover,the helix content as determined decreased by approximately8%, whereas β-sheetcontent displayed apparent increasing for13.6%. The helix transformed into β-sheet.This is a powerful complement and verification to previous experiment conclusion.The SDS-PAGE analysis presented γ subunits of fibrinogen (FgⅠ) were mostvulnerable to oxidative/nitrative modifications induced by ONOO-.3-D fluorescencequenching of the two characteristic absorption peaks of FgⅠ were also observed withthe increasing doses of ONOO-. Based on this, we reported an in-depth study ofnitrotyrosine sites identification of FgⅠand their susceptibility to peroxynitriteutilizing HPLC-ESI-ITMS/MS strategy. It was performed on three enzyme digests.Trypsin, chymotrypsin and Glu-C over digestion were employed and supplemented toensure the tyrosine coverage of100%and high coverage of matched peptides.According to a large dataset, we estimated injury sites of priority (Y96, Y262, Y274and Y363) during database searching. Among them Y262was the common sensitivesites of Glu-C and trypsin digests. The nitration extent of identified nitrotyrosine sitesdecreased with the descending solution concentration of ONOO-. We also detected the effect of chemical modification of tyrosine(Tyr),methionine(Met) and carboxyl(-COOH) on clot formation and nitration of fibrinogen.It is found that Tyr and Met are closely related to the biological activity. NAIapparently accelerated the clotting activity of Fg, while Ch-T showed inhibition.Modifiers such as Ch-T and NAI accelerated the nitration injury of fibrinogen byONOO-when their levels were lower than0.4-0.5mg/mL. The acid environment ofcarboxyl modification by EDC made fibrinogen denaturation, which inhibited theclotting activity and nitrification to some extent.