Copper-catalyzed Protein Oxidation and Its Modulation by Carbon Dioxide

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. 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. When the ability to store and/or handle copper in cells or plasma is overwhelmed, copper is released from its stores via non-copper-dependent oxidative damage (e.g. myeloperoxidase-mediated oxidations) of sulfhydryl groups (1Linder M.C. Mutat. Res. 2001; 475: 141-152Crossref PubMed Scopus (149) Google Scholar) or copper-mediated oxidative damage to biomolecules (2Beshgetoor D. Hambidge M. Am. J. Clin. Nutr. 1998; 67: 1017S-1021SCrossref PubMed Scopus (100) Google Scholar). Indeed, the occurrence of loosely bound copper ions has been reported for a number of clinical samples from pathological conditions involving oxidative stress and inflammation, such as in Wilson disease plasma during an episode of fulminant hepatitis, rheumatoid arthritis synovial fluid, Parkinson disease cerebrospinal fluid, senile plaques of Alzheimer disease (3Gaetke L.M. Chow C.K. Toxicology. 2003; 189: 147-163Crossref PubMed Scopus (1414) Google Scholar, 4Schumann K. Classen H.G. Dieter H.H. Konig J. Multhaup G. Rukgauer M. Summer K.H. Bernhardt J. Biesalski H.K. Eur. J. Clin. Nutr. 2002; 56: 469-483Crossref PubMed Scopus (63) Google Scholar), and blood, aqueous humor, and vitreous bodies of diabetes mellitus patients (5Lin J. Jpn. J. Ophthalmol. 1997; 41: 130-137Crossref PubMed Scopus (28) Google Scholar). Copper-catalyzed oxidations and alteration of tissue components have been implicated in organ damage in many of these pathologies (2Beshgetoor D. Hambidge M. Am. J. Clin. Nutr. 1998; 67: 1017S-1021SCrossref PubMed Scopus (100) Google Scholar, 3Gaetke L.M. Chow C.K. Toxicology. 2003; 189: 147-163Crossref PubMed Scopus (1414) Google Scholar, 4Schumann K. Classen H.G. Dieter H.H. Konig J. Multhaup G. Rukgauer M. Summer K.H. Bernhardt J. Biesalski H.K. Eur. J. Clin. Nutr. 2002; 56: 469-483Crossref PubMed Scopus (63) Google Scholar, 6Stadtman E.R. Levine R.L. Ann. N. Y. Acad. Sci. 2000; 899: 191-208Crossref PubMed Scopus (941) Google Scholar, 7Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar, 8Halliwell B. Whiteman M. Br. J. Pharmacol. 2004; 142: 231-255Crossref PubMed Scopus (1732) Google Scholar, 9Stadtman E.R. Free Radic. Biol. Med. 1990; 9: 315-325Crossref PubMed Scopus (1011) Google Scholar, 10Halliwell B. Gutteridge J.M. Blake D. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1985; 311: 659-671Crossref PubMed Scopus (130) Google Scholar, 11Stadtman E.R. Oliver C.N. J. Biol. Chem. 1991; 266: 2005-2008Abstract Full Text PDF PubMed Google Scholar), and in most of them, a site-specific mechanism of oxidative damage to proteins and other biomolecules has been demonstrated (3Gaetke L.M. Chow C.K. Toxicology. 2003; 189: 147-163Crossref PubMed Scopus (1414) Google Scholar, 7Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar, 11Stadtman E.R. Oliver C.N. J. Biol. Chem. 1991; 266: 2005-2008Abstract Full Text PDF PubMed Google Scholar, 12Stadtman E.R. Methods Enzymol. 1995; 258: 379-393Crossref PubMed Scopus (105) Google Scholar, 13Gutteridge J.M. Biochim. Biophys. Acta. 1986; 869: 119-127Crossref PubMed Scopus (193) Google Scholar, 14Chevion M. Free Radic. Biol. Med. 1988; 5: 27-37Crossref PubMed Scopus (446) Google Scholar, 15Kocha T. Yamaguchi M. Ohtaki H. Fukuda T. Aoyagi T. Biochim. Biophys. Acta. 1997; 1337: 319-326Crossref PubMed Scopus (108) Google Scholar). In addition, (bi)carbonate buffer (BB) 1The abbreviations used are: BB, (bi)carbonate buffer; HSA, human serum albumin; ABTS, 2,2′-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid; BCDS, bathocuproinedisulfonic acid; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DNP, dinitrophenylhydrazone; DTNB, 5,5′-dithiobis(2-nitrobenzoic) acid; DTPA, diethylenetriamine-pentaacetic acid; ELISA, enzyme-linked immunosorbent assay; ESR, electron spin resonance; H2O2, hydrogen peroxide; PB, phosphate buffer. 1The abbreviations used are: BB, (bi)carbonate buffer; HSA, human serum albumin; ABTS, 2,2′-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid; BCDS, bathocuproinedisulfonic acid; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DNP, dinitrophenylhydrazone; DTNB, 5,5′-dithiobis(2-nitrobenzoic) acid; DTPA, diethylenetriamine-pentaacetic acid; ELISA, enzyme-linked immunosorbent assay; ESR, electron spin resonance; H2O2, hydrogen peroxide; PB, phosphate buffer.(CO2, H2CO3, HCO3− and CO3−2) has not only become recognized as an important component in vivo and in cell culture maintenance of acid-base balance but has also been implicated in the modulation of the oxidative chemistry of peroxynitrite (16Augusto O. Bonini M.G. Amanso A.M. Linares E. Santos C.C.X. De Menezes S.L. Free Radic. Biol. Med. 2002; 32: 841-859Crossref PubMed Scopus (459) Google Scholar, 17Pryor W.A. Lemercier J.-N. Zhang H. Uppu R.M. Squadrito L.G. Free Radic. Biol. Med. 1997; 23: 331-338Crossref PubMed Scopus (94) Google Scholar, 18Tien M. Berlett B.S. Levine R.L. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7809-7814Crossref PubMed Scopus (155) Google Scholar) and other reactive oxygen species (19Liochev S.I. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12485-12490Crossref PubMed Scopus (36) Google Scholar, 20Vesela A. Wilhelm J. Physiol. Rev. 2002; 51: 335-339Google Scholar). Thus, (bi)carbonate could affect the mechanism of oxidative damage to proteins by copper-catalyzed oxidations. On the other hand, evidence of the involvement of free radicals in the oxidative damage to proteins by H2O2-induced, copper-catalyzed oxidations has been limited to the detection of protein fragmentation, carbonyl groups, and other end products resulting from oxidative modification of amino acids (8Halliwell B. Whiteman M. Br. J. Pharmacol. 2004; 142: 231-255Crossref PubMed Scopus (1732) Google Scholar, 12Stadtman E.R. Methods Enzymol. 1995; 258: 379-393Crossref PubMed Scopus (105) Google Scholar, 21Stadtman E.R. Annu. Rev. Biochem. 1993; 62: 797-821Crossref PubMed Scopus (1252) Google Scholar, 22Amici A. Levine R.L. Tsai L. Stadtman E.R. J. Biol. Chem. 1989; 264: 3341-3346Abstract Full Text PDF PubMed Google Scholar, 23Stadtman E.R. Berlett B.S. J. Biol. Chem. 1991; 266: 17201-17211Abstract Full Text PDF PubMed Google Scholar, 24Hawkins C.L. Davies M.J. Biochim. Biophys. Acta. 2001; 1504: 196-219Crossref PubMed Scopus (598) Google Scholar, 25Chevion M. Berenshtein E. Stadtman E.R. Free Radic. Res. 2001; 33: 99S-108SGoogle Scholar). To our knowledge, nobody has detected protein radicals in the well known system of human serum albumin (HSA)/Cu(II)/H2O2. If such detection is possible by means of immunospin trapping (26Mason R.P. Free Radic. Biol. Med. 2004; 36: 1214-1223Crossref PubMed Scopus (144) Google Scholar, 27Ramirez D.C. Gomez Mejiba S.E. Mason R.P. Free Radic. Biol. Med. 2005; 38: 201-214Crossref PubMed Scopus (54) Google Scholar) (see Scheme I), it would represent a notable advantage of this technique over the standard ESR techniques for detecting protein radicals, which are compromised by the broad ESR signal of Cu(II) (28Hanna P.M. Mason R.P. Arch. Biochem. Biophys. 1992; 295: 205-213Crossref PubMed Scopus (127) Google Scholar, 29Hanna P.M. Chamulitrat W. Mason R.P. Arch. Biochem. Biophys. 1992; 296: 640-644Crossref PubMed Scopus (109) Google Scholar). Thus, immunospin trapping could be a powerful tool for detecting protein radicals in complex systems such as cells. In the present investigation, to study H2O2-induced, copper-catalyzed protein radicals in functioning cells, we have validated the recently developed immunospin trapping technique with DMPO using the HSA/Cu(II)/H2O2 system as a model. Our results suggest a novel mechanism to explain carbon dioxide-modulated, H2O2-induced, copper-catalyzed oxidation that can help us explain the highly specific DMPO trapping of protein radicals in cells exposed to Cu(II) and H2O2. Reagents—Anhydrous CuCl2 (99.999% purity), ZnCl2 (99.999%), and NaHCO3 (99.7-100.3%) were purchased from Alfa Aesar (Ward Hill, MA). Human serum albumin (HSA, 99.99% purity), carbonate dehydratase, bathocuproinedisulfonic acid acid and were purchased from ABTS, and were from The spin trap DMPO was purchased from by and The DMPO was a of H2O2 was from The H2O2 was using were with to found in phosphate as Free Radic. Biol. Med. 1990; PubMed Scopus Google Scholar). The of the buffer NaHCO3 was to by a Human was in phosphate buffer with we our of a with phosphate buffer to and a of in of albumin bound copper that of the albumin bound copper in M. Arch. Biochem. Biophys. 2004; PubMed Scopus Google Scholar). the of our with H2O2 and DMPO for not protein fragmentation or nitrone by or and ELISA, that our not In addition, it has been that in of of free sulfhydryl groups have formed M. Arch. Biochem. Biophys. 2004; PubMed Scopus Google Scholar). To in our of was with a of by the of phosphate buffer. The in our was to that of the we or not were with in the of H2O2-induced, nitrone was from its of free sulfhydryl groups in and was as Methods Enzymol. PubMed Scopus Google Scholar). samples were with of and of in of in the the of the was and from a and a of sulfhydryl an for to was To the of reduction of sulfhydryl groups by a using E.R. Mason R.P. Chem. Res. 2002; PubMed Scopus Google Scholar) was the from our of or H2O2, and Cu(II) were in the or of DMPO in phosphate in a of were for and by of the H2O2 by of of in phosphate buffer. to Cu(II) or were in phosphate buffer in a of in MA). were for The Cu(II) in the was by H2O2-induced reduction of Cu(II) to Parkinson E. J. J. Biol. Chem. 2000; 5: PubMed Scopus Google Scholar). of were with of and of H2O2 and for by the of of phosphate and the of the complex was to and of with 264.7 cells were as in with serum and before were in to of in of with serum in with to serum with or of BB, and DMPO was added and cells for Cu(II) and H2O2 were added and the for were by the with and the cells were by and with to proteins and cells were in of PB, and ability to was using a a the cell was in of phosphate buffer and by was by using serum as a H2O2-induced, Copper-catalyzed oxidation of by a system Cu(II) H2O2 and of which was added from a NaHCO3 was in phosphate When or was was or The oxidation of was during the first the of H2O2 and the of was using its the nitrone of DMPO was in our and used in the detection of D. Mason R.P. Free Radic. Biol. Med. 2002; 33: PubMed Scopus (105) Google Scholar), D.C. Mason R.P. Free Radic. Biol. Med. 2003; PubMed Scopus Google Scholar, D.C. Mason R.P. 2003; PubMed Scopus Google Scholar, D.C. Mason R.P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and radicals D.C. Gomez Mejiba S.E. Mason R.P. Free Radic. Biol. Med. 2005; 38: 201-214Crossref PubMed Scopus (54) Google Scholar) as nitrone The is from number number and number nitrone were using a standard in as D.C. Gomez Mejiba S.E. Mason R.P. Free Radic. Biol. Med. 2005; 38: 201-214Crossref PubMed Scopus (54) Google Scholar). and were by the were using or the proteins were to a were blocked with a in (bi)carbonate for were with buffer in and exposed to the nitrone a of in buffer for by The was added a of in buffer. for the was by The were detected by to phosphate from for for groups in were using an protein oxidation detection from number with with the and were detected using the system as that used in our that the was used a of H2O2-induced, was with we fragmentation of and and of the to fragmentation was an in carbonyl groups in The of fragmentation and carbonyl formation the of but sites of fragmentation and the site-specific mechanism for oxidative damage to proteins by oxidations E.R. Levine R.L. Ann. N. Y. Acad. Sci. 2000; 899: 191-208Crossref PubMed Scopus (941) Google Scholar, 12Stadtman E.R. Methods Enzymol. 1995; 258: 379-393Crossref PubMed Scopus (105) Google Scholar, 14Chevion M. Free Radic. Biol. Med. 1988; 5: 27-37Crossref PubMed Scopus (446) Google Scholar). When DMPO was in the reaction mixture, we nitrone formation as by nitrone were only HSA, DMPO, and H2O2 were present In addition, damage and of nitrone were prevented in a by or not that H2O2 and of copper were for the of radicals and site-specific fragmentation of HSA. When the was and the reaction of HSA, H2O2, and DMPO was in phosphate the fragmentation of and nitrone increased with not When was with H2O2 for Cu(II) a in site-specific fragmentation of as demonstrated with these conditions and in the of DMPO, Cu(II) nitrone that were by nitrone as and of HSA, that are but radical DMPO-trappable radicals, which are as in the site-specific fragmentation of E.R. Levine R.L. Ann. N. Y. Acad. Sci. 2000; 899: 191-208Crossref PubMed Scopus (941) Google Scholar, 24Hawkins C.L. Davies M.J. Biochim. Biophys. Acta. 2001; 1504: 196-219Crossref PubMed Scopus (598) Google Scholar). When was with DMPO, H2O2, and of that a of Cu(II) as as was to nitrone our the signal in in samples and DMPO was not that our does not In addition, nitrone were detected DMPO was added a of HSA/Cu(II)/H2O2 by the of not that DMPO in real time, a species to a radical that to the DMPO and by Copper-catalyzed and as well as other of free hydroxyl radicals (i.e. and to oxidative fragmentation, a for free hydroxyl radicals chemistry by free copper in our We a H2O2-induced, copper-catalyzed oxidative fragmentation of carbonyl formation and increased of nitrone DMPO of fragmentation and carbonyl formation is with the formation of DMPO of radical in the oxidative fragmentation of HSA, we the that are also and by DMPO to In and are present the as B and and to and H2O2-induced, Copper-catalyzed of Cu(II) to by that of the Cu(II) was in the first When H2O2 was added to the mixture, copper was in the in our the added Cu(II) is bound to and is chemistry resulting from free When the Cu(II) was added before the of H2O2, it nitrone formation of was to such nitrone to a was to the formation was added to the reaction a was in complex Parkinson E. J. J. Biol. Chem. 2000; 5: PubMed Scopus Google Scholar), which was prevented by or not When was with we H2O2-induced, copper-catalyzed oxidative fragmentation, as by an in the as well as a in fragmentation also nitrone these of to a of and the of used in these not H2O2-induced, copper-catalyzed oxidative damage to and that could not from of its sites as was recently W. J. A. J. Biochem. 1998; PubMed Scopus Google Scholar). the free by hydroxyl radical formation in which radicals in a the of amino acids H2O2-induced, copper-catalyzed oxidative fragmentation and nitrone formation to a and not not affect protein fragmentation or of such nitrone and but of (bi)carbonate a of H2O2-induced, nitrone but fragmentation of that the of (bi)carbonate in systems Cu(II) and H2O2 enhanced H2O2-induced, copper-catalyzed oxidation of the results with the enhanced oxidative damage to by the system (bi)carbonate was added The of carbonate to phosphate buffer HCO3− a in the of H2O2-induced, copper-catalyzed of the of or carbonate in phosphate to this system not the effect of (bi)carbonate oxidation these results suggest that free or copper-catalyzed oxidations are enhanced by carbon Copper-catalyzed in the of (bi)carbonate in H2O2-induced, copper-catalyzed oxidations of proteins in RAW 264.7 we exposed to H2O2 and Cu(II) for in phosphate buffer of and the of protein radicals and a of cell were by immunospin trapping and the When cells were with of Cu(II) and H2O2 in phosphate (bi)carbonate a in nitrone was in cell or the system has effect the protein We that (bi)carbonate increased carbonyl groups as demonstrated by of exposed to Cu(II) and H2O2 in phosphate buffer or (bi)carbonate a in nitrone a and specific and protein radical formation in of the in a of cell the well known HSA/Cu(II)/H2O2 we have that DMPO protein radicals, in situ and in real time, functioning cells. We have also the radical formation that are by carbon dioxide immunospin trapping for the detection of radicals, we the of the oxidative damage to by copper-catalyzed oxidations and In the we and pathophysiological of copper-catalyzed protein oxidation has been in many oxidative K. Classen H.G. Dieter H.H. Konig J. Multhaup G. Rukgauer M. Summer K.H. Bernhardt J. Biesalski H.K. Eur. J. Clin. Nutr. 2002; 56: 469-483Crossref PubMed Scopus (63) Google Scholar, 6Stadtman E.R. Levine R.L. Ann. N. Y. Acad. Sci. 2000; 899: 191-208Crossref PubMed Scopus (941) Google Scholar, 11Stadtman E.R. Oliver C.N. J. Biol. Chem. 1991; 266: 2005-2008Abstract Full Text PDF PubMed Google Scholar, 25Chevion M. Berenshtein E. Stadtman E.R. Free Radic. Res. 2001; 33: 99S-108SGoogle Scholar, E.R. Free Radic. Biol. Med. 2002; 33: PubMed Scopus Google Scholar, 1990; PubMed Google Scholar, B. Biol. Google Scholar) and its modulation by of by Copper-catalyzed in suggest that and copper to HSA, as the and The of which Free Radic. Res. 1990; 9: PubMed Scopus Google Scholar, Free Radic. Res. 1989; PubMed Scopus Google Scholar), blocked protein fragmentation and protein radicals the of to the reaction does not affect protein radical our this amino acid is in copper DMPO nitrone The a and an that and sites for the of Cu(II) M. Arch. Biochem. Biophys. 2004; PubMed Scopus Google Scholar, W. J. A. J. Biochem. 1998; PubMed Scopus Google Scholar). and in has recently been using techniques and W. J. A. J. Biochem. 1998; PubMed Scopus Google Scholar). most in the of and of W. J. A. J. Biochem. 1998; PubMed Scopus Google Scholar). Thus, to the chemistry of free copper in our we used of to the copper sites bound to HSA, copper ions can M. Arch. Biochem. Biophys. 2004; PubMed Scopus Google Scholar) to an H2O2-induced in to the protein that be as Cu(II)/.OH (i.e. Scopus Google Scholar), which was not by of free hydroxyl radicals to the of in the protein E.R. Free Radic. Biol. Med. 1990; 9: 315-325Crossref PubMed Scopus (1011) Google Scholar, 14Chevion M. Free Radic. Biol. Med. 1988; 5: 27-37Crossref PubMed Scopus (446) Google Scholar, 15Kocha T. Yamaguchi M. Ohtaki H. Fukuda T. Aoyagi T. Biochim. Biophys. Acta. 1997; 1337: 319-326Crossref PubMed Scopus (108) Google Scholar). In to in this the chemistry of free copper in our system and demonstrated that bound site-specific are in our model. DMPO Copper-catalyzed by in and in before with fragmentation was blocked by DMPO can be explained by the known ability of DMPO to Cu(II) P.M. Chamulitrat W. Mason R.P. Arch. Biochem. Biophys. 1992; 296: 640-644Crossref PubMed Scopus (109) Google as in nitrone were enhanced by results suggest that DMPO site-specific fragmentation, it species in the oxidative fragmentation of HSA, radicals, in situ and in real time, before they can with oxygen (see Scheme it is important to that to radical and can affect the of the protein radical by DMPO a 24Hawkins C.L. Davies M.J. Biochim. Biophys. Acta. 2001; 1504: 196-219Crossref PubMed Scopus (598) Google also Scheme H2O2-induced, Copper-catalyzed but immunospin trapping in the detection of free radical in the HSA/Cu(II)/H2O2 we the effect of (bi)carbonate in the (i.e. and which is present in systems can the mechanism and reaction of the reactive species formed during and conditions of oxidative such as and of Scholar). the of (bi)carbonate in oxidative damage to plasma proteins and cells has more E.R. Berlett B.S. J. Biol. Chem. 1991; 266: 17201-17211Abstract Full Text PDF PubMed Google Scholar, S.I. Fridovich I. Arch. Biochem. Biophys. 2004; PubMed Scopus Google Scholar, S.I. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus Google Scholar, S.I. Fridovich I. Free Radic. Biol. Med. 2004; 36: PubMed Scopus Google Scholar), has been reported to with the H2O2-induced, the to the S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. B. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (100) Google Scholar), which can H. J. B. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (100) Google Scholar, S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) or D.C. Gomez Mejiba S.E. Mason R.P. Free Radic. Biol. Med. 2005; 38: 201-214Crossref PubMed Scopus (54) Google Scholar). We that free Cu(II) and H2O2 can such as not and this reaction is increased by (bi)carbonate and enhanced by carbonate The H2O2-induced, copper-catalyzed oxidation of was not by carbonate or that copper is not a for H2O2-induced, copper-catalyzed oxidation or its of In the of carbonate dehydratase, the of is with a in the of which that a in H2O2-induced, copper-catalyzed of The of oxidation of (bi)carbonate to CO3·− is not to other such as and a or H2O2-induced, to be bound to proteins E.R. Levine R.L. Ann. N. Y. Acad. Sci. 2000; 899: 191-208Crossref PubMed Scopus (941) Google Scholar, 18Tien M. Berlett B.S. Levine R.L. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7809-7814Crossref PubMed Scopus (155) Google Scholar, S.I. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12485-12490Crossref PubMed Scopus (36) Google Scholar, 23Stadtman E.R. Berlett B.S. J. Biol. Chem. 1991; 266: 17201-17211Abstract Full Text PDF PubMed Google Scholar). The of (bi)carbonate to with H2O2 and Cu(II) protein fragmentation, but enhanced nitrone and To explain the the fragmentation and increased radical trapping in the of we propose the formation of a free and the anion are in with H. J. Am. Chem. Soc. 2003; PubMed Scopus Google Scholar, M.G. D.C. O. 2004; PubMed Scopus Google that the of carbonate a in the H2O2-induced, copper-catalyzed oxidation of the in most via the of is a of the H. J. Am. Chem. Soc. 2003; PubMed Scopus Google Scholar). The reduction of by would the CO3·− (16Augusto O. Bonini M.G. Amanso A.M. Linares E. Santos C.C.X. De Menezes S.L. Free Radic. Biol. Med. 2002; 32: 841-859Crossref PubMed Scopus (459) Google Scholar) for the formation of the hydroxyl radical Scopus Google Scholar) or its copper species. The oxidation of the CO3·− radical to the hydroxyl radical-like species is in a more in the of H2O2, oxidation copper M. Arch. Biochem. Biophys. 2004; PubMed Scopus Google Scholar), in the of fragmentation the copper Our and also that H2O2-induced, copper-catalyzed oxidations mediated by a hydroxyl radical-like species fragment the the CO3·− does an important of oxidative damage would suggest that copper-catalyzed reduction of CO3·− produces and protein oxidation (see Scheme but not fragmentation. In the reaction CO3·− (16Augusto O. Bonini M.G. Amanso A.M. Linares E. Santos C.C.X. De Menezes S.L. Free Radic. Biol. Med. 2002; 32: 841-859Crossref PubMed Scopus (459) Google Scholar) and amino acids is Res. 56: PubMed Scopus Google and technique of spin trapping of free radical has a tool in the study of free radical processes in systems R.P. of the Human and Scholar). free radical of (e.g. and M.J. C.L. Free Radic. Biol. Med. 2004; 36: PubMed Scopus Google Scholar) and of radical N. E. J. J. J. B. Free Radic. Biol. Med. 2003; PubMed Scopus (108) Google Scholar) have the detection of protein radicals in functioning cells On the other hand, DMPO is a nitrone spin trap that can cell to in systems M. N. N. in and Scholar). In addition, DMPO to cell N. E. J. J. J. B. Free Radic. Biol. Med. 2003; PubMed Scopus (108) Google Scholar). The of (bi)carbonate to an functioning cells, H2O2, and Cu(II) but increased protein radicals as detected by that our mechanism of reduction by to CO3·− also in cells. that hydroxyl radical-like fragmentation but enhanced DMPO-trappable radical sites in and in functioning a these results would suggest a for as a and/or of H2O2-induced, copper-catalyzed oxidative damage to proteins pathophysiological conditions involving copper-mediated oxidative We Mason and for help in the of the to T. for