Determining the site of spin trapping of the equine myoglobin radical by combined use of EPR, electrophoretic purification, and mass spectrometry.
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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.