title: Tryptophan-14 is the preferred site of DBNBS spin trapping in the self-peroxidation reaction of sperm whale metmyoglobin with a single equivalent of hydrogen peroxide.
abstract: The 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS)-metmyoglobin adduct formed following the horse metmyoglobin-H(2)O(2) reaction has been assigned to both a tyrosyl and a tryptophanyl residue radical. At low H(2)O(2), hyperfine coupling to a (13)C atom in sperm whale metmyoglobin labeled at the tryptophan residues with (13)C allowed the unequivocal assignment of the primary adduct to a tryptophanyl radical. Trapping at Trp-14 of sperm whale myoglobin was indicated by greatly decreased electron paramagnetic resonance (EPR) spectral intensity of the DBNBS adducts of the Trp-14-Phe recombinant proteins. Complex EPR spectra with partially resolved hyperfine splittings from several atoms were obtained by pronase treatment of the DBNBS/*W14F metmyoglobin adducts. The EPR spectra of authentic DBNBS/*Tyr adducts were incubation time-dependent; the late time spectra resembled the spectra of pronase-treated DBNBS/*W14F sperm whale myoglobin adducts, suggesting formation of an unstable tyrosyl radical adduct in the latter proteins. When the H(2)O(2):metmyoglobin ratio was increased to 5:1, the EPR spectrum after pronase treatment supported trapping of a tyrosyl radical, although similar decreases in tryptophan content were detected at H(2)O(2):metmyoglobin ratios of 1:1 and 5:1.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, West Virginia University, Morgantown 26506, USA. mgunther@hsc.wvu.edu
authors.forename: Michael R
authors.initials: MR
authors.lastname: Gunther
authors.forename: Richard A
authors.initials: RA
authors.lastname: Tschirret-Guth
authors.forename: Olivier M
authors.initials: OM
authors.lastname: Lardinois
authors.forename: Paul R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D006736:Horses; D006861:Hydrogen Peroxide / Q000737:chemistry*; D008786:Metmyoglobin / Q000737:chemistry*; D009603:Nitroso Compounds / Q000737:chemistry*; D016877:Oxidants / Q000737:chemistry; D011402:Pronase / Q000737:chemistry; D013045:Species Specificity; D019097:Spin Trapping / Q000379:methods; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry; D014907:Whales
chemical_list: D001557:Benzenesulfonates; D005609:Free Radicals; D009603:Nitroso Compounds; D016877:Oxidants; D008786:Metmyoglobin; D014443:Tyrosine; C048757:3,5-dibromo-4-nitrosobenzenesulfonate; D014364:Tryptophan; D006861:Hydrogen Peroxide; D011402:Pronase