CHARACTERIZATION OF CYTOCHROME-C FREE-RADICAL REACTIONS WITH PEPTIDESBY MASS-SPECTROMETRY

The reactions of horse heart cytochrome c, hydrogen peroxide, and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid with a series of p olypeptides were investigated using mass spectrometry. The mass spectr a obtained from these reactions revealed that after a free radical has been generated on the heme containing protein horse heart cytochrome c, it can be transferred to other biomolecules. Zn addition, the numbe r of free radicals transferred to the target molecule could be determi ned. Recipient peptides/proteins that contained a tyrosine and/or tryp tophan amino acid residue were most susceptible to free radical transf er. Using tandem mass spectrometry, the location of the 3,5-dibromo-4- nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLN K was unequivocally determined to be at the tryptophan residue. We als o demonstrated that the presence of an antioxidant in the reaction mix ture not only inhibits free radical formation on horse heart cytochrom e c, but also interferes with the transfer of the free radical, once i t has been formed on cytochrome c.