Determining the site of spin trapping of the equine myoglobin radical by combined use of EPR, electrophoretic purification, and mass spectrometry.
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.
Harris
Michael N
MN
Toxicology Program, College of Pharmacy, The Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Burchiel
Scott W
SW
Winyard
Paul G
PG
Engen
John R
JR
Mobarak
Charlotte D
CD
Timmins
Graham S
GS
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
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