title: Spin scavenging analysis of myoglobin protein-centered radicals using stable nitroxide radicals: characterization of oxoammonium cation-induced modifications.
abstract: Spin scavenging combined with chromatographic and mass spectrometric procedures can, in principle, be employed to detect and identify protein-based radicals within complex biological matrices. This approach is based on the well-known ability of stable synthetic nitroxide radicals to scavenge carbon-centered radicals, forming stable diamagnetic addition products. Hence, characterization of these addition products would allow for the identification of specific free radicals. In the present work, we have explored the use of the stable nitroxide radical 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) in scavenging protein-based radicals generated in a horse heart metmyoglobin/hydrogen peroxide (metMb/H(2)O(2)) system. Inclusion of a substoichiometric amount of TEMPOL in the metMb/H(2)O(2) system resulted in a complete loss of peroxyl and tyrosyl radical signals and effectively inhibited the formation of oxidatively damaged heme species, as monitored by electron paramagnetic resonance and reversed-phase liquid chromatography. Scavenging of globin radicals by TEMPOL did not lead to the formation of stable diamagnetic addition adducts; in fact, reversed-phase liquid chromatographic studies and oxygen electrode measurements indicated that TEMPOL acts as a catalyst and is recycled in this system. The oxoammonium cation generated in the course of this reaction initiated secondary reactions resulting in the formation of a free carbonyl on the N-terminal Gly-residue of the protein. This oxidative deamination was confirmed through the combined use of reversed-phase liquid chromatographic purification, tandem MS experiments, and chemical analysis (e.g., by use of 2,4-dinitrophenyl hydrazine). The results reveal the pitfalls inherent in using stable nitroxide radicals such as TEMPOL to identify sites of radical formation on hemoproteins.
authors.affiliation: Laboratories of Pharmacology and Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA. lardinois.olivier@gmail.com
authors.forename: Olivier M
authors.initials: OM
authors.lastname: Lardinois
authors.forename: David A
authors.initials: DA
authors.lastname: Maltby
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: Paul R Ortiz
authors.initials: PR
authors.lastname: de Montellano
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Tomer
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
authors.forename: Leesa J
authors.initials: LJ
authors.lastname: Deterding
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D016166:Free Radical Scavengers / Q000378:metabolism; D006736:Horses; D006861:Hydrogen Peroxide / Q000378:metabolism; D008786:Metmyoglobin / Q000378:metabolism; D009211:Myoglobin / Q000378:metabolism*; D009589:Nitrogen Oxides / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D013113:Spin Labels; D053719:Tandem Mass Spectrometry
chemical_list: D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D009211:Myoglobin; D009589:Nitrogen Oxides; D010446:Peptide Fragments; D000644:Quaternary Ammonium Compounds; D013113:Spin Labels; D008786:Metmyoglobin; D006861:Hydrogen Peroxide; C039900:nitroxyl; C001803:tempol