Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization.
5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.
Detweiler
Charles D
CD
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, MD F0-01, Research Triangle Park, NC 27709, USA.
Lardinois
Olivier M
OM
Deterding
Leesa J
LJ
de Montellano
Paul R Ortiz
PR
Tomer
Kenneth B
KB
Mason
Ronald P
RP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D003497:Cyclic N-Oxides / Q000032:analysis / Q000737:chemistry; D019281:Dimerization; D000939:Epitopes / Q000276:immunology; D005609:Free Radicals / Q000032:analysis; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D009589:Nitrogen Oxides / Q000276:immunology; D013113:Spin Labels; D019097:Spin Trapping; D014443:Tyrosine / Q000032:analysis*; D014907:Whales
D000906:Antibodies; D003497:Cyclic N-Oxides; D000939:Epitopes; D005609:Free Radicals; D009211:Myoglobin; D009589:Nitrogen Oxides; D013113:Spin Labels; C015388:nitrones; D014443:Tyrosine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006861:Hydrogen Peroxide