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.

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.