Characterization of cytochrome c free radical reactions with peptides by 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 polypeptides were investigated using mass spectrometry. The mass spectra 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. In addition, the number of free radicals transferred to the target molecule could be determined. Recipient peptides/proteins that contained a tyrosine and/or tryptophan amino acid residue were most susceptible to free radical transfer. Using tandem mass spectrometry, the location of the 3,5-dibromo-4-nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLNK was unequivocally determined to be at the tryptophan residue. We also demonstrated that the presence of an antioxidant in the reaction mixture not only inhibits free radical formation on horse heart cytochrome c, but also interferes with the transfer of the free radical, once it has been formed on cytochrome c.
Deterding
L J
LJ
Laboratory of Structural Biology, NIEHS, Research Triangle Park, North Carolina 27709, USA.
Barr
D P
DP
Mason
R P
RP
Tomer
K B
KB
D000595:Amino Acid Sequence; D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry*; D005609:Free Radicals; D006736:Horses; D006861:Hydrogen Peroxide / Q000737:chemistry; D013058:Mass Spectrometry; D009206:Myocardium / Q000737:chemistry; D009603:Nitroso Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D013113:Spin Labels
D001557:Benzenesulfonates; D003574:Cytochrome c Group; D005609:Free Radicals; D009603:Nitroso Compounds; D010455:Peptides; D013113:Spin Labels; C048757:3,5-dibromo-4-nitrosobenzenesulfonate; D006861:Hydrogen Peroxide