Author(s): Ramirez DC, Mejiba SE, Mason RP
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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.
This article was published in J Biol Chem
and referenced in Enzyme Engineering