Detection and Mapping of Widespread Intermolecular Protein Disulfide Formation during Cardiac Oxidative Stress Using Proteomics with Diagonal Electrophoresis

Abstract

Regulation of protein function by reversible cysteine-targeted oxidation can be achieved by multiple mechanisms, such as S-glutathiolation, S-nitrosylation, sulfenic acid, sulfinic acid, and sulfenyl amide formation, as well as intramolecular disulfide bonding of vicinal thiols. Another cysteine oxidation state with regulatory potential involves the formation of intermolecular protein disulfides. We utilized two-dimensional sequential non-reducing/reducing SDS-PAGE (diagonal electrophoresis) to investigate intermolecular protein disulfide formation in adult cardiac myocytes subjected to a series of interventions (hydrogen peroxide, S-nitroso-N-acetylpenicillamine, doxorubicin, simulated ischemia, or metabolic inhibition) that alter the redox status of the cell. More detailed experiments were undertaken with the thiol-specific oxidant diamide (5 mm), a concentration that induces a mild non-injurious oxidative stress. This increase in cellular oxidation potential caused global intermolecular protein disulfide formation in cytosolic, membrane, and myofilament/cytoskeletal compartments. A large number of proteins that undergo these associations were identified using liquid chromatography-mass spectrometry/mass spectrometry. These associations, which involve metabolic and antioxidant enzymes, structural proteins, signaling molecules, and molecular chaperones, were confirmed by assessing “shifts” on non-reducing immunoblots. The observation of widespread protein-protein disulfides indicates that these oxidative associations are likely to be fundamental in how cells respond to redox changes. Regulation of protein function by reversible cysteine-targeted oxidation can be achieved by multiple mechanisms, such as S-glutathiolation, S-nitrosylation, sulfenic acid, sulfinic acid, and sulfenyl amide formation, as well as intramolecular disulfide bonding of vicinal thiols. Another cysteine oxidation state with regulatory potential involves the formation of intermolecular protein disulfides. We utilized two-dimensional sequential non-reducing/reducing SDS-PAGE (diagonal electrophoresis) to investigate intermolecular protein disulfide formation in adult cardiac myocytes subjected to a series of interventions (hydrogen peroxide, S-nitroso-N-acetylpenicillamine, doxorubicin, simulated ischemia, or metabolic inhibition) that alter the redox status of the cell. More detailed experiments were undertaken with the thiol-specific oxidant diamide (5 mm), a concentration that induces a mild non-injurious oxidative stress. This increase in cellular oxidation potential caused global intermolecular protein disulfide formation in cytosolic, membrane, and myofilament/cytoskeletal compartments. A large number of proteins that undergo these associations were identified using liquid chromatography-mass spectrometry/mass spectrometry. These associations, which involve metabolic and antioxidant enzymes, structural proteins, signaling molecules, and molecular chaperones, were confirmed by assessing “shifts” on non-reducing immunoblots. The observation of widespread protein-protein disulfides indicates that these oxidative associations are likely to be fundamental in how cells respond to redox changes. Oxidants have been implicated in the pathogenesis of most major diseases (1Klein J.A. Ackerman S.L. J. Clin. Investig. 2003; 111: 785-793Crossref PubMed Scopus (459) Google Scholar, 2Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7425) Google Scholar, 3Sahaf B. Heydari K. Herzenberg L.A. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4001-4005Crossref PubMed Scopus (120) Google Scholar, 4Kono H. Rusyn I. Yin M. Gäbele E. Yamashina S. Dikalova A. Kadiiska M.B. Connor H.D. Mason R.P. Segal B.H. Bradford B.U. Holland S.M. Thurman R.G. J. Clin. Investig. 2000; 106: 867-872Crossref PubMed Scopus (429) Google Scholar), including those of the heart (5Griendling K.K. FitzGerald G.A. Circulation. 2003; 108: 1912-1916Crossref PubMed Scopus (789) Google Scholar). However, substantial contemporary evidence is emerging that shows that these species play important regulatory roles in the everyday function of cells and tissues (6Soberman R.J. J. Clin. Investig. 2003; 111: 571-574Crossref PubMed Scopus (61) Google Scholar, 7Finkel T. Curr. Opin Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1014) Google Scholar, 8Stamler J.S. Lamas S. Fang F.F. Cell. 2001; 106: 675-683Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar, 9Rhee S.G. Bae Y.-S. Lee S.-R. Kwon J. Science's STKE. 2000; (www.stke.org/cgi/content/full/OC_sigtrans;2000/53/pe1)PubMed Google Scholar). The roles of oxidants as signaling, or regulatory entities, that allow cell function to be coupled to its redox status is being increasingly established. In terms of abundance, the most important redox couples within cells involve the equilibrium between reduced and oxidized forms of thiols. The status of the cellular reduction potential, which essentially is an index of the thiol redox status, varies during the life history of a cell. For example, the cellular reduction potential has characteristic set points depending on whether tissue is quiescent, proliferating, confluent, differentiating, or apoptotic (10Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar, 11Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1148) Google Scholar). Changes in cellular redox potential to a more oxidizing state can be triggered by a number of processes in healthy tissue, such as metabolism and signaling events. Under basal conditions neurohormonal substances not only activate classical signaling pathways, but also transactivate others that are dependent on the generation of endogenously derived oxidants such as superoxide, hydrogen peroxide, or nitric oxide (12Rhee S.G. Exp. Mol. Med. 1999; 31: 53-59sCrossref PubMed Scopus (575) Google Scholar, 13Mahadev K. Wu X. Zilbering A. Zhu L. Lawrence T.R. Goldstein B.J. J. Biol. Chem. 2001; 276: 48662-48669Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 14Lopez-Ongil S. Senchak V. Saura M. Zaragoza C. Ames M. Ballermann B. Rodriguez-Puyol M. Rodriguez-Puyol D. Lowenstein C.J. J. Biol. Chem. 2000; 275: 26423-26427Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). When the cellular oxidation potential is elevated, protein thiols within the cell become susceptible to modification. A number of reversible protein thiol oxidation states can be produced, including S-thiolated (15Eaton P. Byers H.L. Leeds N. Ward M.A. Shattock M.J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), S-nitrosylated (8Stamler J.S. Lamas S. Fang F.F. Cell. 2001; 106: 675-683Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar), sulfenic acid (PSOH) (16Poole L.B. Karplus P.A. Claiborne A. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 325-347Crossref PubMed Scopus (499) Google Scholar), sulfinic acid (PSO2H) (17Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (801) Google Scholar), sulfenyl amide (18Salmeen A. Andersen J.N. Myers M.P. Meng T.-C. Hinks J.A. Tonks N.K. Barford D. Nature. 2003; 423: 769-773Crossref PubMed Scopus (783) Google Scholar), and reactive oxidized lipid derivatives (19Eaton P. Hearse D.J. Shattock M.J. Cardiovasc. Res. 2001; 52: 294-301Crossref Scopus (31) Google Scholar). Vicinal thiols, which form intramolecular protein disulfides, are also an established mode of cysteine-targeted oxidation that allows regulatory control (20Gitler C. Zarmi B. Kalef E. Anal. Biochem. 1997; 252: 48-55Crossref PubMed Scopus (68) Google Scholar). Another form of protein-cysteine oxidation that is predicted to occur in cells under oxidizing conditions involves the formation of a disulfide bond between proteins. In this study we utilized two-dimensional sequential non-reducing/reducing SDS-PAGE (diagonal electrophoresis) (21Sommer A. Traut R.R. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3946-3950Crossref PubMed Scopus (92) Google Scholar), to separate proteins that form disulfides in myocardial cells under an oxidative burden. These proteins have then been identified using LC-MS/MS, 1The abbreviations used are: LC-MS/MS, liquid chromatographymass spectrometry/mass spectrometry; SNAP, S-nitroso-N-acetylpenicillamine; PKA, protein kinase A; PKC, protein kinase C; ANT, adenine nucleotide translocator. and provide new insights as to how proteins and cells respond to changes in their redox environment. Cell Preparation—Calcium-tolerant ventricular myocytes were isolated from the hearts of male Wistar rats (250–300 g) using a standard collagenase digestion protocol as described previously (22James A.F. Ramsey J.E. Reynolds A.M. Hendry B.M. Shattock M.J. Biochem. Biophys. Res. Commun. 2001; 284: 1048-1055Crossref PubMed Scopus (27) Google Scholar). Animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (43Guide for the Care and Use of Laboratory Animals Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D. C.1996Google Scholar). After isolation, myocytes were kept at room temperature in Tyrode buffer for 3 h after isolation before subsequent treatments. Assessing Interventions That Induce Protein-Protein Disulfides—We screened a series of oxidative interventions for their ability to promote intermolecular protein disulfide bonds. Control or treated cells were pelleted at 500 × g for 0.5 min and resuspended in lysis buffer containing 50 mm Tris-HCl, pH 7.5, 5 mm EGTA, 2 mm EDTA, 100 mm NaF, 0.05% digitonin, 1% Triton X-100, and 100 mm maleimide and left on ice for 5min. Soluble protein was collected by centrifugation (10,000 × g for 2 min) and reconstituted in SDS buffer a but with 100 mm The was then by as described Interventions using this with diamide (5 mm for hydrogen or mm for or mm for or mm for mm for metabolic or simulated was by cells in control buffer with and for was achieved by cells in an buffer of mm mm mm mm mm mm mm pH for or in separate cells were and treated for min with diamide concentration 5 or buffer as a cells were pelleted at 500 × g for 0.5 the was and cells were in isolation This was and the was resuspended in lysis buffer containing 50 mm Tris-HCl, pH 7.5, 5 mm EGTA, 2 mm EDTA, 100 mm NaF, 0.05% digitonin, and 100 mm maleimide and left for 5 min on ice with The were then at × g for 2 and the was kept The was in lysis buffer and then resuspended in lysis buffer with 1% Triton The were and the was kept The Triton was and then resuspended in lysis with to proteins for of these that this protocol was were reconstituted in SDS of containing 100 mm was in to thiol and or thiol oxidation during This has been to as (21Sommer A. Traut R.R. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3946-3950Crossref PubMed Scopus (92) Google Scholar). membrane, or in SDS buffer a from control or were on After non-reducing the containing the proteins was and and on of a large The was in SDS buffer containing 50 mm disulfide between for this proteins that were by a disulfide bond are by and then from to the at their in the Control or were to on large and to that can be between treatments. were with to the proteins and then in that of the and to the left of the and that are only after diamide proteins that oxidative associations diamide were in for using was using the In were with to that was reversible or that molecular were of disulfide bond After were to using a used in these to proteins protein kinase and protein protein adenine nucleotide from protein kinase A regulatory and were used to the oxidized proteins. of digestion with was to published P. T. S. E. M. Anal. Biochem. PubMed Scopus Google Scholar, M. A. T. S. L. T. Nature. PubMed Scopus Google Scholar, A. M. M. Anal. Chem. PubMed Scopus Google Scholar), for with a digestion E. I. L. M. M. I. 2001; PubMed Scopus Google Scholar). The was from the by sequential with 50 mm hydrogen buffer and were reduced with and by with After with hydrogen buffer the were with and at to of at in mm hydrogen for h at and were by sequential with mm hydrogen acid, and The were and in acid for spectrometry. were using a to a were in acid, and was a × 0.5 and for 3 min with acid the the to The was then reduced to the was to the and the were the with an acid The was set to and were on with states of or a of and were The was between and depending on the and of the were and with a were identified by the of to in using was and an of 50 was used in I. I. C. M. M. E. 2002; PubMed Scopus Google Scholar). The for of by which the cysteine in disulfide bond not in the oxidative shows a of which were as of a detailed of interventions that promote disulfide bond a of the collected in these are In with hydrogen mm), mm or mm), mm or mm), mm), or simulated 3 or to increase or disulfide in control In diamide (5 and hydrogen and were at these protein associations, as by or and to the left of the or that of the and to the left of the proteins that protein-protein disulfides. was a for metabolic and to protein but this was with diamide (5 and hydrogen We used isolated adult ventricular myocytes under control conditions or with the thiol oxidant diamide at a concentration of or 100 between the cells in of the and evidence that the cells were In subsequent in which we intermolecular disulfide cells were in 5 mm diamide for this we have that the of oxidative we on the cells in these was diamide caused of 2 that the cells are under an oxidative burden. This is by the that myocytes treated with diamide (5 mm for min) of proteins. The by proteins were was to the that the was dependent on the of a disulfide which the of the is a of a The was prepared from control or isolated ventricular proteins such as 2 of the in control as a under basal However, 2 to the left diamide and a on the which was on the and is by a shows a of a intermolecular protein disulfides in control or in the a number of proteins disulfide also intermolecular protein disulfide in the which is in and proteins. these widespread protein-protein disulfide bond formation, in multiple mild oxidative stress. that of the protein diamide were identified using of The of proteins identified in this is in I. We to a number of the cysteine in disulfide formation, which are identified by However, on the this was not We of of and of the of the to be by that these are in disulfide of proteins that form intermolecular disulfide during oxidative kinase protein kinase protein kinase protein to protein to to A to to A to protein protein to to to to in a new the of the adenine nucleotide as a protein that protein disulfide formation using we under non-reducing or conditions by multiple diamide only a of the of this protein these In we the of the regulatory of which was also identified using we the of this kinase to its molecular disulfide bond We also the of this protein and that the kinase in the diamide and diamide that diamide the of the regulatory of to undergo a molecular which can be by during this molecular in the shows non-reducing which that form associations diamide of the with caused the to be the associations involve intermolecular protein disulfide bonds. these associations, was only to a with and in which a substantial of the disulfide The were also for and which are also to redox thiols and be to form disulfides. However, in to the the not undergo protein in Health and cells are in the reduced redox the of which is dependent on the equilibrium between oxidized and reduced thiols (10Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar, A. Annu. Rev. Biochem. 52: PubMed Scopus Google Scholar). as well as protein thiols, are in and the cellular redox in a more oxidized state under basal including the J.S. J. Cell Biol. 2004; PubMed Scopus Google and the B. Heydari K. Herzenberg L.A. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4001-4005Crossref PubMed Scopus (120) Google Scholar). occur in healthy cells of and (12Rhee S.G. Exp. Mol. Med. 1999; 31: 53-59sCrossref PubMed Scopus (575) Google Scholar, 13Mahadev K. Wu X. Zilbering A. Zhu L. Lawrence T.R. Goldstein B.J. J. Biol. Chem. 2001; 276: 48662-48669Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 14Lopez-Ongil S. Senchak V. Saura M. Zaragoza C. Ames M. Ballermann B. Rodriguez-Puyol M. Rodriguez-Puyol D. Lowenstein C.J. J. Biol. Chem. 2000; 275: 26423-26427Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In oxidative has been implicated in the pathogenesis of including those of the (1Klein J.A. Ackerman S.L. J. Clin. Investig. 2003; 111: 785-793Crossref PubMed Scopus (459) Google Scholar, 2Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7425) Google Scholar, 3Sahaf B. Heydari K. Herzenberg L.A. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4001-4005Crossref PubMed Scopus (120) Google Scholar, 4Kono H. Rusyn I. Yin M. Gäbele E. Yamashina S. Dikalova A. Kadiiska M.B. Connor H.D. Mason R.P. Segal B.H. Bradford B.U. Holland S.M. Thurman R.G. J. Clin. Investig. 2000; 106: 867-872Crossref PubMed Scopus (429) Google Scholar, K.K. FitzGerald G.A. Circulation. 2003; 108: 1912-1916Crossref PubMed Scopus (789) Google Scholar). and of Interventions That Induce Protein-Protein two-dimensional non-reducing/reducing SDS-PAGE (diagonal electrophoresis) has been used previously to investigate proteins that form disulfides with proteins (21Sommer A. Traut R.R. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3946-3950Crossref PubMed Scopus (92) Google Scholar). the nitric oxide to these intermolecular protein disulfides, which we predicted protein or occur also to increase disulfides, but this study was by the that cell the of this which is used in but cardiac oxidative a increase in protein-protein disulfides. In diamide and hydrogen were of these not only to in oxidative but is also an important of cell signaling S.G. Bae Y.-S. Lee S.-R. Kwon J. Science's STKE. 2000; (www.stke.org/cgi/content/full/OC_sigtrans;2000/53/pe1)PubMed Google Scholar), and disulfide bond formation be an important by which to this oxidant are The diamide with reduced with major this is well by H. H. Press, Scholar), and disulfide is from A predicted of disulfide is protein disulfide and is on with the be an important in the formation of protein-protein disulfides, is to the that in this disulfides can also form or sulfenic acid formation in a that involves subsequent reduction by a protein However, the nitric oxide not promote protein-protein disulfide in these that not these events. we the protein-protein disulfide formation diamide in of the isolated cardiac by to the of the and the of proteins in increase in have been achieved using more However, not protein from the the of proteins in The number of protein-protein disulfides was diamide However, a basal of associations was also from of the and be of a of This basal of oxidation also allows for the of to be coupled to interventions that are (10Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar, 11Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1148) Google Scholar). For example, a during is the of to the of the which is an oxidizing B. Heydari K. Herzenberg L.A. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4001-4005Crossref PubMed Scopus (120) Google Scholar). This the oxidation potential at the of which promote the reduction of cell with the potential of a We have a control to a which in and of during or The control allows proteins that form disulfides to be and not with proteins that of the for 2 a of this protein to the left of the diamide with with proteins disulfide formation during oxidative stress. That study has identified a large number of proteins that form intermolecular disulfides. these as associations, we have also used non-reducing SDS-PAGE and with an to the adenine nucleotide a protein to undergo these oxidative associations using also that disulfide were diamide which were with is an of the and be an important for the redox of this which is important in cell during and and also by J. Res. 2003; PubMed Scopus Google Scholar). These to the we have and the for of disulfides. the of these oxidative on the function of the proteins. This a number of of the large number of proteins that in these events. be a major to the of the multiple associations an is likely that the of proteins in that form these are to be This is by the that most of the proteins we identified are structural or metabolic at in cardiac We only identified a signaling proteins including the regulatory of PKA, kinase and the regulatory of the protein which is with redox thiols (16Poole L.B. Karplus P.A. Claiborne A. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 325-347Crossref PubMed Scopus (499) Google Scholar). We previously kinase in isolated hearts subjected to and (15Eaton P. Byers H.L. Leeds N. Ward M.A. Shattock M.J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, P. Shattock M.J. Circulation. 2001; Scholar). However, this and is not in signaling protein When we on non-reducing we that the of this protein to a molecular with its This observation has been described previously C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and for the of this and disulfide associations, which have for the of cardiac by oxidant disulfide bond we to be during cardiac oxidative (15Eaton P. Byers H.L. Leeds N. Ward M.A. Shattock M.J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, P. Shattock M.J. Circulation. 2001; Scholar). We that not have been using of a abundance, and we intermolecular disulfides using We that to a protein disulfide diamide and in using the we not that or disulfides. This is and others (15Eaton P. Byers H.L. Leeds N. Ward M.A. Shattock M.J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, P. Shattock M.J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. M. S. I. M. V. M. E. A. J. E. P. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, C. I. A. Biochem. Biophys. 2002; PubMed Scopus Google proteins to have redox thiols. This observation the potential for proteins with redox thiols to undergo or forms of oxidative which to the of depending on the mode of cysteine In to with a number of or of protein are diamide that in this and This is with these proteins of during conditions and is an that as is likely to a the cellular structural can respond to its redox and be to cellular to redox stress. these a protein with multiple of of this are by in a study by P.A. M. D. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), have disulfide formation in the of a cell subjected to oxidative by hydrogen peroxide, or also used to the major proteins in these oxidative that these from cell the in the of redox proteins are and of the These used oxidant mm mm hydrogen as used in these When used hydrogen more to these but and be is on the concentration of such as hydrogen peroxide, that in with substantial in the concentration used in are of of hydrogen used in this study M.B. 1998; PubMed Google Scholar), and is that these occur in cells to their of A study C. I. J. J. 2004; disulfide between cardiac proteins and and This shows that disulfides occur in a with the that associations of proteins also is that the associations we have not occur to the during However, we that the associations be likely to but involve a of the protein The of these associations is an for and of number of proteins with have been in this study to form intermolecular disulfides oxidative in isolated cardiac A of the potential for of the proteins and is the of this However, be that of the proteins that have been identified as are to have redox thiols. For example, we have identified of as in tissues under oxidative (15Eaton P. Byers H.L. Leeds N. Ward M.A. Shattock M.J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, P. Shattock M.J. N. Acad. Sci. 2002; PubMed Scopus Google Scholar, P. E. M.J. Leeds N. Byers H.L. Ward M.A. J. Shattock M.J. J. 2003; PubMed Google Scholar). A number of the proteins identified were also to be T. Mol. Cell 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is a of the of oxidants as signaling that the function of tissues in a regulatory by the of thiols. of protein-cysteine such as (15Eaton P. Byers H.L. Leeds N. Ward M.A. Shattock M.J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), (8Stamler J.S. Lamas S. Fang F.F. Cell. 2001; 106: 675-683Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar), sulfenic acid (16Poole L.B. Karplus P.A. Claiborne A. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 325-347Crossref PubMed Scopus (499) Google Scholar), sulfinic acid (17Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (801) Google Scholar), and formation (18Salmeen A. Andersen J.N. Myers M.P. Meng T.-C. Hinks J.A. Tonks N.K. Barford D. Nature. 2003; 423: 769-773Crossref PubMed Scopus (783) Google Scholar), are to to be important in redox This study shows that intermolecular protein disulfide formation is a widespread a proteins, the potential of these oxidative associations in the redox of cell