title: Copper-catalyzed protein oxidation and its modulation by carbon dioxide: enhancement of protein radicals in cells.
abstract: It is well known that hydrogen peroxide (H2O2)-induced copper-catalyzed fragmentation of proteins follows a site-specific oxidative mechanism mediated by hydroxyl radical-like species (i.e. Cu(I)O, Cu(II)/*OH or Cu(III)) that ends in increased carbonyl formation and protein fragmentation. We have found that the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) prevented such processes by trapping human serum albumin (HSA)-centered radicals, in situ and in real time, before they reacted with oxygen. When (bi)carbonate (CO2, H2CO3, HCO3- and CO3(-2)) was added to the reaction mixture, it blocked fragmentation mediated by hydroxyl radical-like species but enhanced DMPO-trappable radical sites in HSA. In the past, this effect would have been explained by oxidation of (bi)carbonate to a carbonate radical anion (CO3*) by a bound hydroxyl radical-like species. We now propose that the CO3* radical is formed by the reduction of HOOCO2- (a complex of H2O2 with CO2) by the protein-Cu(I) complex. CO3* diffuses and produces more DMPO-trappable radical sites but does not fragment HSA. We were also able, for the first time, to detect discrete but highly specific H2O2-induced copper-catalyzed CO3*-mediated induction of DMPO-trappable protein radicals in functioning RAW 264.7 macrophages. We conclude that carbon dioxide modulates H2O2-induced copper-catalyzed oxidative damage to proteins by preventing site-specific fragmentation and enhancing DMPO-trappable protein radicals in functioning cells. The pathophysiological significance of our findings is discussed.
authors.affiliation: Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. ramirez1@niehs.nih.gov
authors.forename: Dario C
authors.initials: DC
authors.lastname: Ramirez
authors.forename: Sandra E Gomez
authors.initials: SE
authors.lastname: Mejiba
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D000818:Animals; D002245:Carbon Dioxide / Q000494:pharmacology*; D002384:Catalysis; D002460:Cell Line; D003300:Copper; D003497:Cyclic N-Oxides / Q000494:pharmacology; D005609:Free Radicals; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D008264:Macrophages / Q000166:cytology; D051379:Mice; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism*
chemical_list: D003497:Cyclic N-Oxides; D005609:Free Radicals; D011506:Proteins; D012709:Serum Albumin; D002245:Carbon Dioxide; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D003300:Copper; D006861:Hydrogen Peroxide