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Reaction of human myoglobin and peroxynitrite: Characterizing biomarkers for myoglobin-derived oxidative stress

TitleReaction of human myoglobin and peroxynitrite: Characterizing biomarkers for myoglobin-derived oxidative stress
Publication TypeJournal Article
Year of Publication2001
AuthorsWitting, PK, Mauk, AG, Douglas, DJ, Stocker, R
JournalBiochemical and Biophysical Research Communications
Volume286
Pagination352-356
Date PublishedAug
Type of ArticleArticle
ISBN Number0006-291X
KeywordsARRHYTHMIAS, free radical, GENERATION, HYDROGEN-PEROXIDE, ischaemia reperfusion injury, MECHANISMS, MYOGLOBIN, NITRATION, NITRIC-OXIDE, nitro-tyrosine, peroxynitrite, protein-thiyl radical, radicals, REPERFUSION INJURY, SITE-DIRECTED MUTAGENESIS, SUPEROXIDE, SYSTEMS
Abstract

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. (C) 2001 Academic Press.

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