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• W2078355435 abstract "Background: Tyrosyl radicals react with superoxide to form bicyclic hydroperoxides that contains an α-β unsaturated carbonyl.Results: GSH undergoes Michael addition with Tyr hydroperoxides in peptides and myoglobin.Conclusion: Proteins that form hydroperoxides through radical-mediated oxidation should readily form GSH-Tyr adducts.Significance: This is a novel mechanism of protein-glutathione adduct formation, and similar chemistry could result in Cys-Tyr cross-linking of proteins during oxidative stress. Background: Tyrosyl radicals react with superoxide to form bicyclic hydroperoxides that contains an α-β unsaturated carbonyl. Results: GSH undergoes Michael addition with Tyr hydroperoxides in peptides and myoglobin. Conclusion: Proteins that form hydroperoxides through radical-mediated oxidation should readily form GSH-Tyr adducts. Significance: This is a novel mechanism of protein-glutathione adduct formation, and similar chemistry could result in Cys-Tyr cross-linking of proteins during oxidative stress. Conjugation of glutathione to oxidized tyrosine residues in peptides and proteins.Journal of Biological ChemistryVol. 290Issue 2PreviewVOLUME 287 (2012) PAGES 26068–26076 Full-Text PDF Open Access Proteins are critical biological targets for reactive oxygen species, and post-translational protein modification is a major mechanism for oxidative damage. It is also a regulatory mechanism for redox signaling and in many cases is structurally important for achieving correct polypeptide folding. Cys, Met, Trp, and Tyr residues are the most oxidant-sensitive, and a wide range of reversible and irreversible modifications have been characterized. One common modification is glutathionylation (1.Schuppe I. Moldéus P. Cotgreave I.A. Protein-specific S-thiolation in human endothelial cells during oxidative stress.Biochem. Pharmacol. 1992; 44: 1757-1764Crossref PubMed Scopus (60) Google Scholar, 2.Gallogly M.M. Mieyal J.J. Mechanisms of reversible protein glutathionylation in redox signaling and oxidative stress.Curr. Opin. Pharmacol. 2007; 7: 381-391Crossref PubMed Scopus (375) Google Scholar, 3.Ghezzi P. Regulation of protein function by glutathionylation.Free Radic. Res. 2005; 39: 573-580Crossref PubMed Scopus (227) Google Scholar, 4.Dalle-Donne I. Rossi R. Colombo G. Giustarini D. Milzani A. Protein S-glutathionylation. A regulatory device from bacteria to humans.Trends Biochem. Sci. 2009; 34: 85-96Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 5.Hill B.G. Bhatnagar A. Protein S-glutathiolation. Redox-sensitive regulation of protein function.J. Mol. Cell Cardiol. 2012; 52: 559-567Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Numerous thiol proteins have been shown to form mixed disulfides with GSH. This reversible process, termed S-glutathionylation, is a means for regulating their activity and is proposed to be one of the mechanisms involved in redox signaling. Glutathionylation through a carbon-sulfur bond can also occur if protein oxidation gives rise to electrophilic sites that could form adducts by Michael addition. The objective of this study was to determine whether tyrosine oxidation to its hydroperoxide could give rise to glutathionylation via this mechanism. Tyr residues are particularly sensitive to one-electron (radical-mediated) oxidation to radicals. These radicals can combine to form dityrosine, which is an important cross-link in some structural proteins as well as a useful biomarker of oxidative stress (6.Heinecke J.W. Tyrosyl radical production by myeloperoxidase. A phagocyte pathway for lipid peroxidation and dityrosine cross-linking of proteins.Toxicology. 2002; 177: 11-22Crossref PubMed Scopus (100) Google Scholar). However, tyrosyl radicals react with superoxide even faster than with each other. This is the prevalent reaction under conditions where both radicals are generated (7.Jin F. Leitich J. von Sonntag C. The superoxide radical reacts with tyrosine-derived phenoxyl radicals by addition rather than by electron transfer.J. Chem. Soc. Perkin Trans. I. 1993; 15: 1583-1588Crossref Scopus (137) Google Scholar, 8.Jonsson M. Lind T. Reitberger T.E. Eriksen T.E. Merenyi G. Free radical combination reactions involving phenoxyl radicals.J. Phys. Chem. 1993; 97: 8229-8233Crossref Scopus (110) Google Scholar, 9.Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides.Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (91) Google Scholar, 10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), such as when tyrosine is oxidized by stimulated neutrophils (11.Winterbourn C.C. Pichorner H. Kettle A.J. Myeloperoxidase-dependent generation of a tyrosine peroxide by neutrophils.Arch. Biochem. Biophys. 1997; 338: 15-21Crossref PubMed Scopus (65) Google Scholar, 12.Nagy P. Kettle A.J. Winterbourn C.C. Neutrophil-mediated oxidation of enkephalins via myeloperoxidase-dependent addition of superoxide.Free Radic. Biol. Med. 2010; 49: 792-799Crossref PubMed Scopus (20) Google Scholar) or in the presence of GSH (13.Pichorner H. Metodiewa D. Winterbourn C.C. Generation of superoxide and tyrosine peroxide as a result of tyrosyl radical scavenging by glutathione.Arch. Biochem. Biophys. 1995; 323: 429-437Crossref PubMed Scopus (71) Google Scholar). The reaction between superoxide and Tyr radicals proceeds either by electron transfer (repair), which reduces the Tyr radical back to Tyr and generates oxygen, or by addition to give a hydroperoxide. When the tyrosine has a free terminal amine, the hydroperoxide is the predominant product (7.Jin F. Leitich J. von Sonntag C. The superoxide radical reacts with tyrosine-derived phenoxyl radicals by addition rather than by electron transfer.J. Chem. Soc. Perkin Trans. I. 1993; 15: 1583-1588Crossref Scopus (137) Google Scholar, 9.Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides.Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (91) Google Scholar). Addition can theoretically occur at the ortho (as shown in Scheme 1, reaction 1) or para position of the Tyr. The repair reaction is more favored when there is no free amino group, but some hydroperoxide is formed in this case too (10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). An alternative route to tyrosine hydroperoxides is the reaction of tyrosyl peptides or proteins with singlet oxygen (14.Wright A. Bubb W.A. Hawkins C.L. Davies M.J. Singlet oxygen-mediated protein oxidation. Evidence for the formation of reactive side chain peroxides on tyrosine residues.Photochem. Photobiol. 2002; 76: 35-46Crossref PubMed Scopus (183) Google Scholar). Hydroperoxide formation on N-terminal Tyr residues is followed by conjugate addition of the Tyr nitrogen to the phenol ring (reaction 2), destroying its aromatic character and generating a bicyclic product (II). The product is less well characterized when there is no free N terminus. Our evidence favors the equivalent of (II) with conjugation to the amide nitrogen (10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14.Wright A. Bubb W.A. Hawkins C.L. Davies M.J. Singlet oxygen-mediated protein oxidation. Evidence for the formation of reactive side chain peroxides on tyrosine residues.Photochem. Photobiol. 2002; 76: 35-46Crossref PubMed Scopus (183) Google Scholar, 15.Saito I. Chujo Y. Shimazu H. Yamane M. Matsuura T. Nonenzymic oxidation of p-hydroxyphenylpyruvic acid with singlet oxygen to homogentisic acid. A model for the action of p-hydroxyphenylpyruvate hydroxylase.J. Am. Chem. Soc. 1975; 97: 5272-5277Crossref PubMed Scopus (50) Google Scholar), but the unconjugated species (I) is also possible. The hydroperoxides readily undergo reduction or hydrolysis (reaction 3) to give the more stable hydroxyl derivative (III) (7.Jin F. Leitich J. von Sonntag C. The superoxide radical reacts with tyrosine-derived phenoxyl radicals by addition rather than by electron transfer.J. Chem. Soc. Perkin Trans. I. 1993; 15: 1583-1588Crossref Scopus (137) Google Scholar). Alternatively, if a Met residue is in the vicinity, Tyr hydroxide is produced through internal transfer of one of the oxygens to give methionine sulfoxide (10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The conjugate addition product (III) still contains an α-β unsaturated carbonyl group so could in theory undergo further Michael addition to thiols and other nucleophiles. This would result in a conjugate with GSH (Scheme 1, reaction 4), and the reaction is a potential mechanism for cross-linking between Tyr and Cys residues in proteins. We have investigated whether this is the case for glutathionylation using small Tyr-containing peptides as well as sperm whale myoglobin, which we have previously shown to form a tyrosyl hydroperoxide derivative when treated with superoxide and hydrogen peroxide (16.Das A.B. Nagy P. Abbott H.F. Winterbourn C.C. Kettle A.J. Reactions of superoxide with the myoglobin tyrosyl radical.Free Radic. Biol. Med. 2010; 48: 1540-1547Crossref PubMed Scopus (25) Google Scholar). We show that GSH reduces the peptide hydroperoxide to hydroxide derivatives (Tyr hydroxide), which readily and efficiently form conjugates. We propose that this could be a physiological mechanism for protein glutathionylation or cross-linking. Tyr-Gly Met-enkephalin, Leu-enkephalin, sperm whale myoglobin, and other reagents and enzymes were purchased from Sigma unless otherwise indicated. Stock solutions of xanthine oxidase (XO) 3The abbreviations used are: XOxanthine oxidaseHRPhorseradish peroxidaseSRMselected reaction monitoringTyr-hydroxidetyrosine hydroxideYG-hydroxideTyr-Gly hydroxide. were prepared by dilution of the ammonium sulfate suspension with 50 mm phosphate buffer, pH 7.4, and spinning through a G25 Sephadex column. Enzyme and acetaldehyde solutions were prepared daily and stored on ice. The custom-synthesized peptide corresponding to the C-terminal end of myoglobin with the sequence ELGYQG was ordered from GenScript (Piscataway, NJ). xanthine oxidase horseradish peroxidase selected reaction monitoring tyrosine hydroxide Tyr-Gly hydroxide. Peptide hydroperoxides were prepared as in Nagy et al. (10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) by treating the Tyr-containing peptide (typically 1–5 mm) in 50 mm sodium phosphate buffer, pH 7.4, containing 50 μm diethylenetriaminepenta-acetic acid and 3 or 10 mm acetaldehyde with 1–3 aliquots (over 30–90 min at 22 °C) of horseradish peroxidase (HRP; final concentration 0.5 μm) and XO (∼0.004 units/ml to give a superoxide generation rate of ∼10 μm/min). The concentration of the HRP stock was determined from its absorbance at 403 nm (ϵ = 112 mm−1cm−1). Superoxide production by XO was measured separately as cytochrome c reduction. The reaction was stopped by adding catalase (10–20 μg/ml), decreasing the volume to half using a vacuum concentrator, then removing the enzymes using a 10- or 50-kDa cut off Amicon Ultra Free-MC Biomax Polysulfone filter (Millipore, Bedford, MA). For ELGYQG, a similar protocol was used except that XO (typically 0.0006 units/ml; superoxide generation 1.5 μm/min) was added to a solution containing 0.2 mm peptide, 2.5 mm acetaldehyde, and 140 nm HRP (16.Das A.B. Nagy P. Abbott H.F. Winterbourn C.C. Kettle A.J. Reactions of superoxide with the myoglobin tyrosyl radical.Free Radic. Biol. Med. 2010; 48: 1540-1547Crossref PubMed Scopus (25) Google Scholar). Reactions were run for 30 min at 22 °C. Tyrosyl hydroperoxide derivatives were either reduced to the corresponding hydroxides by incubating with methionine (typically 0.7 mm) overnight at 20 °C or added directly to GSH (which also performs this reduction). For product analysis, the solutions were treated with GSH (2 mm in pH 7.4 phosphate buffer containing diethylenetriaminepenta-acetic acid). After 1–2 h, they were either analyzed immediately by liquid chromatography-mass spectrometry (LC/MS) or stored at −80 °C. For the experiment using purified Tyr-Gly hydroxide (YG-hydroxide), it was purified from the starting material by liquid chromatography using the LC/MS system. On reinjection of the collected peak, only the YG-hydroxide species were detected. YG-hydroxide was prepared by reducing the hydroperoxide with methionine as above. LC/MS analysis confirmed that no YG-hydroperoxide remained. Phe (internal standard, final concentration 20 μm) plus an equal volume of a GSH solution in the same phosphate buffer was added, and after controlled times at 25 °C samples were injected into the LC/MS system. Controls without GSH were also run at intervals during the analysis period. For each sample, the peak integral for YG-hydroxide was calculated and expressed relative to the Phe peak (m/z = 166). GSH (m/z = 308), which was present in excess, was also monitored to ensure that it was not depleted over the course of the reaction. The loss of YG-hydroxide over time was followed at different GSH concentrations. Sigma Plot was used to fit the kinetic traces to an exponential decay (y = y0 + ae−bt) equation. Sperm whale myoglobin (50 μm in phosphate buffer containing diethylenetriaminepenta-acetic acid) was reacted with XO (typically 0.004 units/ml) plus acetaldehyde (1 mm) for 30 min at 22 °C. Total production of H2O2 over the period was ∼100 μm. It has previously been shown that a superoxide-tyrosyl radical addition product is formed on the myoglobin under these conditions (16.Das A.B. Nagy P. Abbott H.F. Winterbourn C.C. Kettle A.J. Reactions of superoxide with the myoglobin tyrosyl radical.Free Radic. Biol. Med. 2010; 48: 1540-1547Crossref PubMed Scopus (25) Google Scholar). The reaction mixture was then incubated overnight with or without GSH, then treated with 20 mm sodium borohydride (NaBH4) for 30 min in the dark. Reactions were stopped, and heme was extracted from the myoglobin by adding 9 volumes of ice-cold acidified acetone (1% HCl v/v) while vortexing. Precipitated protein was separated, resuspended in 100 mm Tris-HCl buffer, pH 8.5, and incubated with a 50:1 substrate:trypsin ratio for 16 h at 37 °C. Digestion was stopped by the addition of 0.1% formic acid followed by injection of samples onto the LC/MS column. Most of the analyses were performed by liquid chromatography-electrospray ionization (ESI)-mass spectrometry and LC-ESI-MS/MS using a Thermo Finnigan LCQ Deca XP Plus ion trap mass spectrometer (San Jose, CA) in positive ion mode coupled to a Surveyor HPLC system and PDA detector under previously described conditions (10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Data were analyzed using Finnigan Xcalibur, Thermo Finnigan Qual Browser1.3, and HighChem Mass Frontier 3.0 programs. Identity of the peaks was established on the basis of characteristic MS/MS fragmentation patterns. Fragment ions of ELGYQG were assigned based on the Roepstorff-Fohlman nomenclature. Some of the analyses of ELGYQG products and analysis of tryptic digests of myoglobin were performed with an Applied Biosystems 4000QTrap coupled to an Applied Biosystems HPLC system (Concord, Ontario, Canada). A Jupiter 4 μm Proteo 90A C12 150 × 2.0 mm column (Phenomenex, CA) set to 40 °C was used, and separation was achieved using an appropriate gradient of water and acetonitrile containing 0.1% formic acid. All samples were analyzed in the positive ion mode. The electrospray needle was held at 400 °C. Nitrogen was both the curtain and collision gas. The ion spray was 5 kV, and the declustering potential was 40 V. Samples were analyzed by selected ion monitoring, selected reaction monitoring (SRM), or recording the total ion chromatograms. MS/MS experiments were performed using helium as collision gas. Data acquisition and analysis were performed using Analyst 1.4.2 (AB Sciex). Tyr hydroperoxide formation from Tyr radicals and superoxide requires the simultaneous generation of both radicals. This can be achieved using XO/acetaldehyde to generate superoxide and H2O2 plus a peroxidase to catalyze oxidation of the Tyr by the H2O2 (11.Winterbourn C.C. Pichorner H. Kettle A.J. Myeloperoxidase-dependent generation of a tyrosine peroxide by neutrophils.Arch. Biochem. Biophys. 1997; 338: 15-21Crossref PubMed Scopus (65) Google Scholar). An alternative is to generate Tyr radicals in the presence of GSH (13.Pichorner H. Metodiewa D. Winterbourn C.C. Generation of superoxide and tyrosine peroxide as a result of tyrosyl radical scavenging by glutathione.Arch. Biochem. Biophys. 1995; 323: 429-437Crossref PubMed Scopus (71) Google Scholar). In the latter case the glutathionyl radical is formed; this reacts with more GSH to give the glutathione disulfide anion radical (), and superoxide is generated in the reaction of with oxygen. When we used the GSH system with YG and examined the products by LC/MS, we observed the hydroperoxide, but it was short lived. Isomers of YG-hydroxide were formed, but these also disappeared over time, and a peak corresponding to an increase in mass of 307 Da was observed (data not shown). This is consistent with previous studies showing that Tyr hydroperoxides are reduced to hydroxides by GSH (11.Winterbourn C.C. Pichorner H. Kettle A.J. Myeloperoxidase-dependent generation of a tyrosine peroxide by neutrophils.Arch. Biochem. Biophys. 1997; 338: 15-21Crossref PubMed Scopus (65) Google Scholar, 17.Morgan P.E. Dean R.T. Davies M.J. Protective mechanisms against peptide and protein peroxides generated by singlet oxygen.Free Radic. Biol. Med. 2004; 36: 484-496Crossref PubMed Scopus (76) Google Scholar) and also suggested that the hydroxides react with GSH. These reactions were further investigated by adding GSH to preformed YG-hydroxide. YG-OH was prepared by treating the dipeptide with the XO/HRP system then reduction of the hydroperoxide with methionine. As shown in Fig. 1a, LC/MS with selected ion monitoring of the reaction mixture at m/z of 255 (the theoretical value for the hydroxide; III) showed two isomeric peaks. The two peaks had very similar fragmentation patterns that are consistent with the isomeric structures of III, as established previously (10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). After treatment with 5 mm GSH, these peaks disappeared, and a peak with two unresolved shoulders appeared (Fig. 1b). This peak showed the same mass throughout, corresponding to the addition of GSH (+307 Da) to YG-hydroxide. This mass is consistent with a Michael addition reaction, and the fragmentation pattern (Fig. 1c) indicates that the mass increase is due to the addition of GSH to Tyr-hydroxide. The unresolved peaks are likely to correspond to structural isomers, more of which are theoretically possible for the GSH adduct than for the hydroxide alone. The comparable total areas of the peaks in Figs. 1, a and b, suggest efficient conversion. To exclude the possibility that any of the reagents used to prepare YG-hydroxide were involved in the formation of the GSH adduct, YG-hydroxide was purified and reacted with GSH. Analysis by direct infusion mass spectrometry again showed conversion to the same GSH adduct as in Fig. 1c, indicating a direct reaction between GSH and YG-hydroxide. Gly-Tyr, Met-enkephalin, and Leu-enkephalin all form radical addition products with superoxide (10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12.Nagy P. Kettle A.J. Winterbourn C.C. Neutrophil-mediated oxidation of enkephalins via myeloperoxidase-dependent addition of superoxide.Free Radic. Biol. Med. 2010; 49: 792-799Crossref PubMed Scopus (20) Google Scholar). The hydroxide products from these peptides also reacted with GSH to form adducts. LC/MS analysis of the products gave masses corresponding to the addition of GSH, and the major fragments identify them as Michael addition products equivalent to that for YG. The fragmentation patterns indicate that the GSH is localized on the modified Tyr-hydroxide residues (see Table 1 and Fig. 2). As shown for YG-hydroxide, reaction with Cys formed the equivalent Cys conjugate.TABLE 1Identification of Michael addition products formed from superoxide-modified tyrosyl peptidesPeptidem/z [peptide-OH +H]+Thiol (mass)Observed product m/z [peptide-OH + thiol+H]+Major fragments m/zDaYG255Cys (122)376237, 255, 358GY255GSH (307)562415, 433, 544Met-enkephalin YGGFMaThe initial Tyr-hydroperoxide spontaneously undergoes intramolecular oxygen transfer to give a dioxide with the extra oxygens on the Met and modified Tyr residues Nagy et al. (10).606 (dioxide)GSH (307)913606, 784Leu-enkephalin YGGFL572GSH (307)879750, 804a The initial Tyr-hydroperoxide spontaneously undergoes intramolecular oxygen transfer to give a dioxide with the extra oxygens on the Met and modified Tyr residues Nagy et al. (10.Nagy P. Kettle A.J. Winterbourn C.C. Superoxide-mediated formation of tyrosine hydroperoxides and methionine sulfoxide in peptides through radical addition and intramolecular oxygen transfer.J. Biol. Chem. 2009; 284: 14723-14733Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Open table in a new tab Kinetic experiments were carried out with YG-hydroxide using LC/MS to follow its consumption over time. Loss of both hydroxide isomers (shown in Fig. 3a for the peak eluting at 4 min) occurred over minutes or hours depending on the GSH concentration. GSH conjugate peaks increased in parallel with the decrease of the hydroxides, but product formation was not analyzed kinetically. At the lower GSH concentrations, the reactions did not go to completion, suggesting that reaction 4 (Scheme 1) should be represented as an equilibrium, As all kinetic experiments were conducted under pseudo first order conditions using an excess of GSH, the GSH concentration can be treated as constant, and Equation 1 was simplified to,K4=k4k-4=[IV][III][GSH](Eq. 1) K4'=k4'k-4=[IV][III](Eq. 2) where K4′ = K4[GSH] and k4′ = k4[GSH]. Kinetic analysis needs to take into account both the forward and the reverse reactions of an equilibrium, and for reaction 4, the following rate law can be derived (18.Espenson J.H. Chemical Kinetics and Reaction Mechanisms. McGraw-Hill Inc., New York1995: 46-69Google Scholar), where [III]e is the concentration of YG-hydroxide at equilibrium. Integration of Equation 3 gives,d[III]dt=(k4'+k-4)([III]-[III]e)(Eq. 3) [III]t=[III]e+([III]0-[III]e)e-(k4'+k-4)t(Eq. 4) where [III]t is the concentration of YG-hydroxide at a given time point. Therefore, from the y = y0 + ae−bt) equation that was used to fit the experimental data sets, the following parameters were calculated for each kinetic run.K4'=a/y0(Eq. 5) K4'=b/(1+y0/a)(Eq. 6) For each YG-hydroxide isomer, both K4′ and k4′ gave linear dependences on GSH concentration (Fig. 3, b and c). From the slopes in Fig. 3b, the obtained second order rate constants (k4) for the forward reactions are 11.8 ± 0.7 m−1min−1 for the isomer eluting at 4 min and 9.2 ± 0.2 m−1min−1 for the 11-min isomer. The respective equilibrium constants (K4) calculated from the plots in Fig. 3c are (7.5 ± 1.2) × 103 and (21 ± 4) × 103 m−1. From Equation 1, these give reverse rate constants (k−4) of 0.0016 and 0.0004 min−1, respectively. As further evidence of reversibility, the addition of excess Cys to the GSH conjugate of YG-hydroxide resulted in replacement of the conjugated GSH with Cys. The product had similar chromatographic mobility, mass, and MS/MS fragmentation patterns as the YG-hydroxide-Cys adduct (see Table 1). Formation of this product was accompanied by a decrease of ∼60% in the integral of the YG-hydroxide-GSH peaks. We next investigated whether GSH could conjugate to superoxide-modified Tyr on a protein. Sperm whale myoglobin was chosen because it reacts with H2O2 to form a radical on Tyr-151 (19.Lardinois O.M. Ortiz de Montellano P.R. Intra- and intermolecular transfers of protein radicals in the reactions of sperm whale myoglobin with hydrogen peroxide.J. Biol. Chem. 2003; 278: 36214-36226Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and this radical can combine with superoxide to form an addition product (16.Das A.B. Nagy P. Abbott H.F. Winterbourn C.C. Kettle A.J. Reactions of superoxide with the myoglobin tyrosyl radical.Free Radic. Biol. Med. 2010; 48: 1540-1547Crossref PubMed Scopus (25) Google Scholar). The synthetic peptide ELGYQG, an analog of the tryptic peptide 148–153 that contains Tyr-151, was initially examined. As observed previously (16.Das A.B. Nagy P. Abbott H.F. Winterbourn C.C. Kettle A.J. Reactions of superoxide with the myoglobin tyrosyl radical.Free Radic. Biol. Med. 2010; 48: 1540-1547Crossref PubMed Scopus (25) Google Scholar), treatment with the HRP/XO system produced a hydroperoxide (not shown), which decomposed at 37 °C overnight and gave rise to peaks of m/z 682.3 corresponding to the hydroxide (Fig. 4a). The integrals of the LC/MS peaks of the hydroxides disappeared when the reaction mixture was treated with GSH along with the formation of several peaks of m/z 495.2. This corresponds in mass to the double-charged ion of the hydroxide plus 307 Da (Fig. 4b). The MS/MS spectra of all the peaks were similar and indicate that they are isomers of the glutathionylated form of the ELGYQG peptide (Fig. 4, c and d). The fact that GSH is conjugated to the modified Tyr-hydroxide residue is clear from the mass increase of 307 Da from the corresponding y2 to y3 fragments. Myoglobin was then reacted with the XO system (16.Das A.B. Nagy P. Abbott H.F. Winterbourn C.C. Kettle A.J. Reactions of superoxide with the myoglobin tyrosyl radical.Free Radic. Biol. Med. 2010; 48: 1540-1547Crossref PubMed Scopus (25) Google Scholar) to generate the Tyr-hydroxide derivative. When this sample was treated with GSH and digested with trypsin, only minimal conversion of the ELGYQG hydroxide species to its GSH conjugate was detected (not shown). However, the tryptic digest protocol involved removal of the residual GSH by acetone precipitation and incubating the protein with trypsin in Tris-HCl buffer. It is possible that the GSH adduct could have formed on the protein, but subsequent incubation in the absence of GSH could have shifted the equilibrium toward decomposition and regeneration of the hydroxide derivative. This was confirmed by adding NaBH4 after the GSH, on the rationale that it would reduce the carbonyl bond present on the bicyclic ring (IV) and stabilize the adduct species. Initial experiments were carried out with the GSH adduct of the synthetic peptide. The addition of NaBH4 led to the disappearance of the m/z 495.2 peaks (shown in Fig. 4b) and formation of major and several minor peaks at m/z 496.2 (Fig. 5a). It is important to note that these are double-charged species, so their singly charged counterparts will be 2 Da apart. These species all had a similar fragmentation pattern that is consistent with the 2-Da increase corresponding to reduction of the carbonyl group of the modified Tyr residue (Fig. 5, b and c). After blocking the residual GSH with iodoacetamide, the stability of the GSH adducts was compared. In contrast to the time-dependent disappearance of the adduct (Fig. 6a) and recovery of the hydroxide (Fig. 6b) observed with non-reduced species, there was no loss of the reduced GSH adduct. Therefore, reduction of the GSH adduct with NaBH4 was successful in producing a stable, well characterized species.FIGURE 6Time course for dissociation of the GSH adduct of ELGYQG and prevention by NaBH4 reduction. Loss of GSH adduct without (●) or with NaBH4 reduction (○) is shown. a, the GSH adduct was prepared with and without NaBH4 reduction as in Fig. 4 then reacted with iodoacetamide (5 mm). Samples were removed at intervals and analyzed by LC/MS with SRM at m/z 495.2/449.3 for the non-reduced and m/z 496.2/450.3 for the reduced GSH addu" @default.
• W2078355435 created "2016-06-24" @default.
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• W2078355435 creator A5043961236 @default.
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• W2078355435 date "2012-07-01" @default.
• W2078355435 modified "2023-09-29" @default.
• W2078355435 title "Conjugation of Glutathione to Oxidized Tyrosine Residues in Peptides and Proteins" @default.
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