title: Determining the site of spin trapping of the equine myoglobin radical by combined use of EPR, electrophoretic purification, and mass spectrometry.
abstract: Although myoglobin protein radicals are thought important intermediates in peroxide-induced toxicity, the site of spin trapping of this radical in equine myoglobin using the trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) is unclear. We have combined EPR, electrophoretic adduct purification, and mass spectrometry approaches to unambiguously determine the site of trapping to be Tyr-103 and suggest that reports of trapping at Trp-7 or Trp-14 may be due to nonradical addition to proteolytically derived Trp-containing peptides with DBNBS. The technique developed here of combining electrophoretic separation of DBNBS adducts with MS of resultant peptides will also allow proteomic-like approaches to determining identities and sites of radical formation and translocation on complex mixtures of proteins.
authors.affiliation: Toxicology Program, College of Pharmacy, The Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Harris
authors.forename: Scott W
authors.initials: SW
authors.lastname: Burchiel
authors.forename: Paul G
authors.initials: PG
authors.lastname: Winyard
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
authors.forename: Charlotte D
authors.initials: CD
authors.lastname: Mobarak
authors.forename: Graham S
authors.initials: GS
authors.lastname: Timmins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009603:Nitroso Compounds / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013113:Spin Labels; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D001557:Benzenesulfonates; D005609:Free Radicals; D009211:Myoglobin; D009603:Nitroso Compounds; D013113:Spin Labels; D014443:Tyrosine; C048757:3,5-dibromo-4-nitrosobenzenesulfonate; D006861:Hydrogen Peroxide; D014357:Trypsin