Reaction of human myoglobin and peroxynitrite: characterizing biomarkers for myoglobin-derived oxidative stress.
Mixtures of human myoglobin (Mb) (or the Y103F variant of human Mb), authentic peroxynitrite (ONOO(-), ONOO(-):protein 2 mol/mol), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave radicals adducts at cysteine-110 (DMPO-C110) that are detected directly by electron paramagnetic magnetic spectroscopy (EPR). DMPO-C110 was detected exclusively over a range of DMPO concentrations (DMPO:protein ratios 25-100 mol/mol). Treatment of human Mb (or Y103F Mb) with the ONOO(-) generator 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium (SIN-1) chloride (ONOO(-):protein 5 mol/mol) yielded a cross-linked Mb dimer as judged by SDS-PAGE analyses. Addition of DMPO or carbonate effectively eliminated the cross-linked product. Mass analyses of samples containing human Mb (or Y103F Mb), carbonate, and ONOO(-) indicated that nitration occurs exclusively at Y103. Thus, reaction of human Mb and ONOO(-) yields specific products that depend on the presence or absence of physiological concentrations of carbonate. These products may serve as biomarkers for the participation of Mb-derived radicals in the oxidative damage associated with myocardial reperfusion injury.
Witting
P K
PK
Heart Research Institute, 145 Missenden Road, Camperdown Sydney, New South Wales, 2050, Australia. p.witting@hri.org.au
Mauk
A G
AG
Douglas
D J
DJ
Stocker
R
R
D015415:Biomarkers / Q000032:analysis; D003497:Cyclic N-Oxides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000032:analysis; D006801:Humans; D008981:Molsidomine / Q000031:analogs & derivatives / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D015428:Myocardial Reperfusion Injury / Q000175:diagnosis*; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism*
D015415:Biomarkers; D003497:Cyclic N-Oxides; D005609:Free Radicals; D009211:Myoglobin; D009566:Nitrates; D017382:Reactive Oxygen Species; C039642:peroxynitric acid; C002385:linsidomine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D008981:Molsidomine