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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• 作者：Michael N. Harris ; Scott W. Burchiel ; Paul G. Winyard ; John R. Engen ; Charlotte D. Mobarak ; and Graham S. Timmins
• 刊名：Chemical Research in Toxicology
• 出版年：2002
• 出版时间：December 2002
• 年：2002
• 卷：15
• 期：12
• 页码：1589 - 1594
• 全文大小：121K
• 年卷期：v.15,no.12(December 2002)
• ISSN：1520-5010

文摘

Although myoglobin protein radicals are thought important intermediates in peroxide-inducedtoxicity, 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 adductpurification, and mass spectrometry approaches to unambiguously determine the site oftrapping to be Tyr-103 and suggest that reports of trapping at Trp-7 or Trp-14 may be due tononradical addition to proteolytically derived Trp-containing peptides with DBNBS. Thetechnique developed here of combining electrophoretic separation of DBNBS adducts with MSof resultant peptides will also allow proteomic-like approaches to determining identities andsites of radical formation and translocation on complex mixtures of proteins.