Mechanism of S-Nitrosothiol Formation and Degradation Mediated by Copper Ions

Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu2+, but not Fe3+, induces in the presence of NO a fastS-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu+ is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu2+, the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu2+ inS-nitrosothiol formation with the known destabilizing effect of Cu+, providing a unique simple picture where the redox state of copper determines either the NO release fromS-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation. Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu2+, but not Fe3+, induces in the presence of NO a fastS-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu+ is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu2+, the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu2+ inS-nitrosothiol formation with the known destabilizing effect of Cu+, providing a unique simple picture where the redox state of copper determines either the NO release fromS-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation. S-Nitrosothiols (RS-NOs) 1The abbreviations used are:RS-NOS-nitrosothiolBSAbovine serum albuminBSA-SNOS-nitroso-BSAGSHglutathioneGSNOS-nitrosoglutathioneGSSGdimeric glutathioneHbhuman hemoglobinDTNB5,5′-dithiobis-2-nitrobenzoic acidNEMN-ethylmaleimidePMBp-hydroxymercuribenzoic acid 1The abbreviations used are:RS-NOS-nitrosothiolBSAbovine serum albuminBSA-SNOS-nitroso-BSAGSHglutathioneGSNOS-nitrosoglutathioneGSSGdimeric glutathioneHbhuman hemoglobinDTNB5,5′-dithiobis-2-nitrobenzoic acidNEMN-ethylmaleimidePMBp-hydroxymercuribenzoic acid have a variety of biological activities, which are mostly attributed to their ability to release NO (1Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4129) Google Scholar, 2Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 3Stamler J.S. Jia L. Eu J.P. McMahon T.J. Demchenko I.T. Bonaventura J. Gernert K. Piantadosi C.A. Science. 1997; 276: 2034-2037Crossref PubMed Scopus (940) Google Scholar). RS-NOs are not only synthesized and administered clinically (2Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar) but are also produced endogenously. Stamler et al. (4Stamler J.S. Jaraki O. Osborne J. Simon D.J. Keaney J. Vita J. Singel D. Valeri C.R. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7674-7677Crossref PubMed Scopus (1122) Google Scholar) reported that human plasma contains ∼7 μmRS-NOs, mostly as S-nitroso-albumin, a level unexpectedly high as the basal cellular NO level is in the low nanomolar range (5Malinski T. Taha Z. Nature. 1992; 358: 676-678Crossref PubMed Scopus (1022) Google Scholar,6Shibuki K. Okada D. Nature. 1991; 349: 326-328Crossref PubMed Scopus (791) Google Scholar). Thus, RS-NOs are considered as NO pools buffering the level of NO, which may be targeted at different sites (7Pietraforte D. Mallozzi C. Scorza G. Minetti M. Biochemistry. 1995; 34: 7177-7185Crossref PubMed Scopus (75) Google Scholar). RS-NOs are also reported to be involved in the trans-S-nitrosation of proteins by transferring the NO+ moiety (8Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2430) Google Scholar, 9Simon D.I. Mullins M.E. Jia L. Gaston B. Singel D.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4736-4741Crossref PubMed Scopus (184) Google Scholar), a process suggested to be a reversible post-translational modification regulating the activity of enzymes and receptors (3Stamler J.S. Jia L. Eu J.P. McMahon T.J. Demchenko I.T. Bonaventura J. Gernert K. Piantadosi C.A. Science. 1997; 276: 2034-2037Crossref PubMed Scopus (940) Google Scholar, 10Stamler J.S. Simon D.I. Osborne J.A. Mullins M.E. Jaraki O. Michel T. Singel D.J. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 444-448Crossref PubMed Scopus (1286) Google Scholar, 11Xu L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-237Crossref PubMed Scopus (855) Google Scholar). S-nitrosothiol bovine serum albumin S-nitroso-BSA glutathione S-nitrosoglutathione dimeric glutathione human hemoglobin 5,5′-dithiobis-2-nitrobenzoic acid N-ethylmaleimide p-hydroxymercuribenzoic acid S-nitrosothiol bovine serum albumin S-nitroso-BSA glutathione S-nitrosoglutathione dimeric glutathione human hemoglobin 5,5′-dithiobis-2-nitrobenzoic acid N-ethylmaleimide p-hydroxymercuribenzoic acid The degradation of RS-NOs depends on many factors including light, pH, metal ions and the presence of reductants (e.g. ascorbate or thiols) (12Askew S.C. Barnett D.J. McAninly J. Williams D.L.H. J. Chem. Soc. Perkin Trans. 1995; 2: 741-745Crossref Scopus (151) Google Scholar, 13Pfeiffer S. Schrammel A. Schmidt K. Mayer B. Anal. Biochem. 1998; 258: 68-73Crossref PubMed Scopus (42) Google Scholar, 14Scorza G. Pietraforte D. Minetti M. Free Radical Biol. Med. 1997; 22: 633-642Crossref PubMed Scopus (117) Google Scholar, 15Kashiba-Iwatsuki M. Kitoh K. Kasahara E., Yu, H. Nisikawa M. Matsuo M. Inoue M. J. Biochem. ( Tokyo ). 1997; 122: 1208-1214Crossref PubMed Scopus (62) Google Scholar, 16Singh S.P. Wishnok J.S. Keshive M. Deen W.M. Tannenbaum S.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14428-14433Crossref PubMed Scopus (296) Google Scholar); RS-NOs are quite stable in pure buffer solutions (hours; Ref. 17Singh R.J. Hogg N. Joseph J. Kalyanaraman B. J. Biol. Chem. 1996; 271: 18596-18603Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar), but they decompose rapidly (within seconds) upon irradiation with visible light or by transition metal catalysis (18Gorren A.C.F. Schrammel A. Schmidt K. Mayer B. Arch. Biochem. Biophys. 1996; 330: 219-228Crossref PubMed Scopus (162) Google Scholar). Therefore, their stability and reactivity in biological systems can hardly be predicted. RS-NO formation is even less understood than degradation. At neutral pH, NO does not react directly with glutathione (GSH) to form S-nitrosoglutathione (GSNO), since only a slow redox reaction forming N2O and dimeric glutathione (GSSG) occurs (k′ = 4.8 × 10−4 s−1at 5 mm GSH; Ref. 19Hogg N. Singh R.S. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (246) Google Scholar). Under aerobic conditions the auto-oxidation of NO generates N2O3(k ∼6 × 106m−2 s−1; Ref. 20Ford P.C. Wink D.A. Stanbury D.M. FEBS Lett. 1993; 326: 1-3Crossref PubMed Scopus (391) Google Scholar), which is able to S-nitrosate thiols. N2O3 reacts with both thiols and water, the two reactions proceeding atk = 0.1 - 1 × 105m−1 s−1 (21Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar), for low molecular weight thiols, and k ∼ 30m−1 s−1 (21Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar), respectively. It is worth noticing that the reaction with water efficiently competes with direct thiol nitrosation by N2O3, due to the large molar excess of water over thiols. NAD+ substituting oxygen for the electron acceptor can also accelerate the reaction of NO with thiols (22Gow A.J. Buerk D.G. Ischiropoulos H. J. Biol. Chem. 1997; 272: 2841-2845Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Several authors also suggested thatS-nitrosation of thiols occurs by reaction with nitrosonium ions (NO+) formed either via metal-catalyzed oxidation of NO or via dinitrosyl-iron-cysteine complexes (8Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2430) Google Scholar, 21Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 23Vanin A.F. Malenkova I.V. Serezhenkov V.A. Nitric Oxide. 1997; 1: 191-203Crossref PubMed Scopus (170) Google Scholar, 24Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar); efficiency and physiological relevance of these reactions remain unclear. In this study we have examined by spectroscopic and amperometric techniques the interaction of NO and thiols in the presence of cupric and ferric ions. Experiments have been carried out using the small tripeptide GSH (low millimolar amounts in the cell), bovine serum albumin (BSA, which is the most abundant plasma protein), and human hemoglobin (Hb). BSA and GSH both bear only one reduced cysteine per molecule (Cys-34 in BSA; Refs. 25Min He X. Carter D.C. Nature. 1992; 358: 209-215Crossref PubMed Scopus (3408) Google Scholar and 26Kashiba-Iwatsuki M. Miyamoto M. Inoue M. Arch. Biochem. Biophys. 1997; 345: 237-242Crossref PubMed Scopus (37) Google Scholar), but, as shown below, they display in the presence of Cu2+ a very different reactivity with NO. Hb has been reported to undergoS-nitrosation, the reaction occurring at the level of Cys-β93 (27Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1454) Google Scholar). We found that Cu2+, but not Fe3+, catalyzes the rapid S-nitrosation of BSA with a stoichiometry of ∼1 SNO/BSA, and of Hb with a stoichiometry dependent on the derivative used. GSH showed under similar conditions no reaction with NO, probably because of fast thiol dimerization. In contrast to other reports (8Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2430) Google Scholar, 21Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 23Vanin A.F. Malenkova I.V. Serezhenkov V.A. Nitric Oxide. 1997; 1: 191-203Crossref PubMed Scopus (170) Google Scholar, 24Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), our evidence does not indicate a role for NO+ in Cu2+-inducedS-nitrosation. As this Cu2+-mediated reaction is fast, selective for thiols, and efficient, it may be relevant for RS-NO formation in vitro. GSH was obtained from Roche Molecular Biochemicals; HgCl2, Cu(II)SO4, and Cu(I)Cl from Merck; DTNB, EDTA, NEM, PMB, neocuproine, and BSA (catalog no. A-2153) from Sigma Aldrich. Hb was purified according to Ref. 28Rossi-Fanelli A. Antonini E. Caputo A. J. Biol. Chem. 1961; 236: 165-168Abstract Full Text PDF Google Scholar. When necessary Hb was treated with thiol blocking reagents: 10-fold excess Hg2+ or 3–5-fold excess PMB over Hb of BSA and GSH used in water, and the concentration of thiols was by the Arch. Biochem. Biophys. PubMed Scopus Google Scholar); GSH and BSA The concentration of thiols in BSA is in with reports G. Pietraforte D. Minetti M. Free Radical Biol. Med. 1997; 22: 633-642Crossref PubMed Scopus (117) Google Scholar) and probably due to M. M. Keaney Loscalzo J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, B. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). BSA are therefore with to thiol concentration of Hb to the NO was from and from by a solutions of NO by water with the NO at 1 this 0.1 mm NO at Free Radical Biol. Med. 1998; PubMed Scopus Google Scholar), as also by of reduced G. A. M. Biochem. Biophys. 1998; PubMed Scopus Google Scholar). of by of in of mm and the from light with and carried out at using a 1 Cu2+ NO to buffer either GSH or 1 mm was to Cu2+, by the of was since the that is upon reaction of thiols with DTNB, is by The at was stable formation the of DTNB, thiol concentration was using the = and in with Arch. Biochem. Biophys. PubMed Scopus Google Scholar). have been using buffer in a of BSA in 0.1 was for with and NO. 1 mm was and a the of Cu2+, by a in the of NO, the at the of formed N. Singh R.S. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (246) Google Scholar). to the concentration of thiols, was and the at was the of NO was using a NO to a with a The was with of water, the NO concentration in the was under at was 0.1 and spectroscopic have been to the interaction of metal NO, thiols, and The amperometric method is for NO no to or the of thiols and the formation of RS-NOs carried out in provide since the formation of RS-NOs the of both NO and thiols. a role of Cu2+ in the formation of GSH and BSA NO, Cu2+, or NO the concentration of thiols was by the The in 1 NO and Cu2+ the thiols of BSA to = no effect and thiols, = of thiols was in the presence of 1 1 (18Gorren A.C.F. Schrammel A. Schmidt K. Mayer B. Arch. Biochem. Biophys. 1996; 330: 219-228Crossref PubMed Scopus (162) Google Scholar), that ions are formed the reaction of Cu2+ with BSA; since Cu+ is known to degradation of RS-NOs (12Askew S.C. Barnett D.J. McAninly J. Williams D.L.H. J. Chem. Soc. Perkin Trans. 1995; 2: 741-745Crossref Scopus (151) Google Scholar, A.C.F. Schrammel A. Schmidt K. Mayer B. Arch. Biochem. Biophys. 1996; 330: 219-228Crossref PubMed Scopus (162) Google Scholar), the the formation of of GSH with excess NO a small effect on the thiol concentration in contrast to thiol concentration upon of Cu2+ 1 In the presence of both NO and Cu2+, thiols from carried out with ferric of cupric ions showed no of thiols using either BSA or GSH both with and NO and a role of in the S-nitrosation of thiols (e.g. formation of similar have been also under to the to a effect of in these NO was in excess over Under these of thiols of of the presence or of 1 When the NO concentration not by of either BSA or Cu2+ and the other the of BSA to a of both NO and Cu2+ was with of NO the reaction was fast by the of the T. S. 1996; PubMed Google Scholar), and a stoichiometry of of Cu+ all the NO by BSA this direct evidence for the formation of as Cu+ with the release of NO (12Askew S.C. Barnett D.J. McAninly J. Williams D.L.H. J. Chem. Soc. Perkin Trans. 1995; 2: 741-745Crossref Scopus (151) Google A.C.F. Schrammel A. Schmidt K. Mayer B. Arch. Biochem. Biophys. 1996; 330: 219-228Crossref PubMed Scopus (162) Google Scholar). The was in the presence of small amounts of neocuproine, formed in the reaction of BSA with Cu2+, under these conditions upon of 1 from and the reaction was upon of excess of Experiments in the presence of of of Cu2+, to a but of NO from of BSA not can probably be by the formation of a with high for NO. this reaction, which can be in of concentration was at 1 have been carried out with either or a of the of NO under aerobic the of NO by BSA was similar in both not The that has no effect on the reaction of S-nitrosation of BSA also the of thiol in the reaction of BSA and NO, the was with excess of either or of BSA to the reaction NO and Cu2+, no of NO was of BSA to a NO and of Cu2+ not in a of NO only NO the of of was Therefore, with the spectroscopic a activity of in formation can be with the spectroscopic 1 of GSH to a of NO and Cu2+ showed no of NO other from can the Cu2+-mediated S-nitrosation of Hb was by the amperometric NO As shown in Hb either in the or was to the NO a fast NO was to NO to the in a stoichiometry of Hb with thiol blocking of Cu2+, a rapid of NO was which was attributed to RS-NO prevented by Hb with thiol blocking and and by of Cu+ and The stoichiometry was using and using The different can be that the reactivity of Cys-β93 can on the state of Hb (27Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1454) Google Scholar). formed of BSA with Cu2+ and NO was RS-NOs are by a with a at N. Singh R.S. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (246) Google Scholar). to the low of all by of of BSA at different with amounts of NO and Cu2+ showed at which was not in the of The of the in to for at = in with the = at D.J. H. N. B. B. FEBS Lett. 345: PubMed Scopus Google Scholar) and very to = for N. Singh R.S. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (246) Google Scholar). When 1 mm was to the BSA and Cu2+ of NO, formation was prevented not BSA was with amounts of NO in the presence of excess was to cupric ions and both the formation of and the concentration of thiols the of As shown in upon the NO the of cysteine of BSA the concentration The reaction is very even at a of the reaction has reports the concentration of thiols the aerobic of BSA to of NO and the concentration of Cu2+ As shown in the to a of no of was the of Cu2+ to a of to of the of Cu2+ was even to a of The carried out in the presence of showed a similar but a of the reaction efficiency the of is at a of these that formation and stability of on the of NO, Cu2+, and Cu+ formed in the reaction BSA and We found in the presence of NO, cupric ions can a fast S-nitrosation of both BSA and used as ferric ions are in this is on amperometric and spectroscopic showed that NO from in a in the presence of both BSA and Cu2+, but not in the presence of either BSA or Cu2+ formation is by the NO release upon of Cu+ to the reaction which is known to the of RS-NOs with a release of NO (12Askew S.C. Barnett D.J. McAninly J. Williams D.L.H. J. Chem. Soc. Perkin Trans. 1995; 2: 741-745Crossref Scopus (151) Google Scholar, A.C.F. Schrammel A. Schmidt K. Mayer B. Arch. Biochem. Biophys. 1996; 330: 219-228Crossref PubMed Scopus (162) Google Scholar). carried out with Hb S-nitrosation with stoichiometry dependent on the Hb derivative or showed that the of BSA to the was with NO and Cu2+, but not with either one of the two of was by the of the at in where of BSA with NO and Cu2+ the at was = in with the D.J. H. N. B. B. FEBS Lett. 345: PubMed Scopus Google Scholar). of amounts of NO to BSA in the presence of Cu2+, we a of the thiol concentration and a of the of The of thiol and for the of thiols The of in carried out in the or presence of was the also on the that no reaction was the aerobic of BSA with NO the of we can a role of oxygen in this process The concentration of BSA in low to efficiently with water in the reaction with N2O3 formed in the auto-oxidation reaction of NO (21Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). the evidence the mechanism shown in for the of The of of BSA Cu2+ forming a which reacts with NO to mechanism is as both Cu2+ and BSA are for the reaction with it is to that in this The formation of a is also suggested by the in the presence of excess the formation of a by a and K. Anal. Chem. Scopus Google Scholar), was in only upon of NO to BSA and Cu2+, that the is stable and the release of Cu+ may occur only the of NO to form The mechanism presented in is as the reaction of NO with thiols does not directly as suggested (8Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2430) Google Scholar, 21Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 23Vanin A.F. Malenkova I.V. Serezhenkov V.A. Nitric Oxide. 1997; 1: 191-203Crossref PubMed Scopus (170) Google Scholar, 24Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) and reported NO+ is known to react with water to form in the presence of Cu2+ the in be NO+ and NO rapidly this was not in the amperometric reported in which even in the presence of a excess of Cu2+, no of NO a that out a role of NO+ in RS-NO formation at at micromolar amounts of also to be by the which the of copper ions by thiols than by NO it is known that Cu2+ GSH and cysteine D. C. S. G. Arch. Biochem. Biophys. Scopus Google from Refs. Free Radical Biol. Med. 1998; PubMed Scopus Google Scholar of and Scholar in a The of as as the of with GSH or are described in the Cu2+ is by the of glutathione probably forming a J. Chem. Soc. PubMed Scopus Google Scholar, K. K. H. J. Chem. Soc. Scopus Google Scholar); both the and the involved redox reaction from Ref. J. Chem. Soc. PubMed Scopus Google Scholar) are to be very fast s−1 and s−1; Ref. A.C.F. Schrammel A. Schmidt K. Mayer B. Arch. Biochem. Biophys. 1996; 330: 219-228Crossref PubMed Scopus (162) Google Scholar). general including the reactions in and the of is reported in also a simple for the different of the glutathione and the BSA or Hb with Cu2+ and NO. The of GSH to the NO and Cu2+ was not by of NO, the of GSH with a spectroscopic to of thiols 1 can be that Cu2+ induces of the reaction in with the formation of from our the reaction of NO with glutathione may be fast, it is by the is not as the thiols in the are in very from Ref. J. Chem. Soc. PubMed Scopus Google Scholar). In contrast, the of the is since and formation is by et al. R. J.S. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) reported that of BSA is only under and oxidation to or acid only occurs in the presence of as with these of BSA with Cu2+ we not a of the thiol fast oxidation of and of study of the role of Cu2+ in the reaction of thiols with NO. The shown in indicate that Cu2+ in a of The formation of depends on the of Cu2+ and to a of no of thiols was The of Cu2+ to a of thiols. is with BSA two copper as also suggested by et al. B. Biochemistry. 1995; 34: PubMed Scopus Google Scholar); the is and the is involved in the reaction with NO. et al. B. Biochemistry. 1995; 34: PubMed Scopus Google Scholar) suggested that Cu2+ to BSA with high to the of and of and with to Therefore, Cu2+ the high and react with in the presence of NO. the thiol with Hg2+ or prevented the reaction with NO. The formation of and is probably to the presence of Cu+ formed in the reaction of Cu2+ with BSA and As reported Cu+ is known to the of RS-NOs NO and (12Askew S.C. Barnett D.J. McAninly J. Williams D.L.H. J. Chem. Soc. Perkin Trans. 1995; 2: 741-745Crossref Scopus (151) Google A.C.F. Schrammel A. Schmidt K. Mayer B. Arch. Biochem. Biophys. 1996; 330: 219-228Crossref PubMed Scopus (162) Google Scholar). In formation of Cu+ therefore decompose the The of the RS-NO therefore on the the formation and As in the presence of neocuproine, a for the efficiency of formation As by the Cu2+-mediated nitrosation of by the release of NO by Cu+, the mechanism presented can also be as a method for in vitro S-nitrosation of It over is by with low molecular weight RS-NOs or reaction they have to be used in large excess over the and to the of a is As the reactions described are efficient, formation of RS-NO is even at micromolar of under these conditions the reaction can be directly either or the of a it may be of to also on a role of copper ions in the and NO most of Cu2+ is of plasma Ref. B. Biochem. J. Scopus Google Scholar), and albumin B. Biochem. J. Scopus Google Scholar); the and concentration of copper ions is very low D. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). we a physiological relevance of the reactions it be to a role for copper ions in in S-nitrosation. We and for We also K. for of the