Glutathione Sulfonic Acid Research Articles

Study on Chemical Modifications of Glutathione by Cold Atmospheric Pressure Plasma (Cap) Operated in Air in the Presence of Fe(II) and Fe(III) Complexes

Cold atmospheric pressure plasma is an attractive new research area in clinical trials to treat skin diseases. However, the principles of plasma modification of biomolecules in aqueous solutions remain elusive. It is intriguing how reactive oxygen and nitrogen species (RONS) produced by plasma interact on a molecular level in a biological environment. Previously, we identified the chemical effects of dielectric barrier discharges (DBD) on the glutathione (GSH) and glutathione disulphide (GSSG) molecules as the most important redox pair in organisms responsible for detoxification of intracellular reactive species. However, in the human body there are also present redox-active metals such as iron, which is the most abundant transition metal in healthy humans. In the present study, the time-dependent chemical modifications on GSH and GSSG in the presence of iron(II) and iron(III) complexes caused by a dielectric barrier discharge (DBD) under ambient conditions were investigated by IR spectroscopy, mass spectrometry and High Performance Liquid Chromatography (HPLC). HPLC chromatograms revealed one clean peak after treatment of both GSH and GSSH with the dielectric barrier discharge (DBD) plasma, which corresponded to glutathione sulfonic acid GSO3H. The ESI-MS measurements confirmed the presence of glutathione sulfonic acid. In our experiments, involving either iron(II) or iron(III) complexes, glutathione sulfonic acid GSO3H appeared as the main oxidation product. This is in sharp contrast to GSH/GSSG treatment with DBD plasma in the absence of metal ions, which gave a wild mixture of products. Also interesting, no nitrosylation of GSH/GSSG was oberved in the presence of iron complexes, which seems to indicate a preferential oxygen activation chemistry by this transition metal ion.

Capillary electrophoresis with mass spectrometric detection for separation of S-nitrosoglutathione and its decomposition products: a deeper insight into the decomposition pathways.

S-Nitrosoglutathione (GSNO) is a very important biomolecule that has crucial functions in many physiological and physiopathological processes. GSNO acts as NO donor and is a candidate for future medicines. This work describes, for the first time, the separation and the detection of GSNO and its decomposition products using capillary electrophoresis coupled to mass spectrometry (CE-MS). The separation was performed in slightly alkaline medium (pH8.5) under positive-ionization MS detection. The identification of three byproducts of GSNO was formally performed for the first time: oxidized glutathione (GSSG), glutathione sulfinic acid (GSO2H), and glutathione sulfonic acid (GSO3H). GSO2H and GSO3H are known to have important biological activity, including inhibition of the glutathione transferase family of enzymes which are responsible for the elimination of many mutagenic, carcinogenic, and pharmacologically active molecules. We observed, after the ageing of GSNO in the solid state, that the proportion of both GSSG and GSO3H increases whereas that of GSO2H decreases. These results enabled us to propose an oxidation scheme explaining the formation of such products.

Quantitation of glutathione and its oxidation products in erythrocytes by multiple-label stable-isotope dilution

A multiple-label stable isotope dilution assay for quantifying glutathione (GSH), glutathione disulfide (GSSG), and glutathione sulfonic acid in erythrocytes was developed. As the internal standards, [13C3,15N]glutathione, [13C4,15N2]glutathione disulfide, and [13C3,15N]glutathione sulfonic acid were used. Analytes and internal standards were detected by LC–MS/MS after derivatization of GSH with iodoacetic acid and dansylation of all compounds under study. The calibration functions for all analytes relative to their respective isotopologic standards revealed slopes close to 1.0 and negligible intercepts. As various labelings of the standards for GSH and GSSG were used, their simultaneous quantitation was possible, although GSH was partly oxidized to its disulfide during analysis. The degree of this artifact formation of GSSG was calculated from the abundance of the mixed disulfide formed from unlabeled GSH and its respective standard. Thus, the detected GSSG amount could be corrected for the artifact amount. In this way, the amount of GSSG in erythrocytes was found to be less than 0.5% of the GSH concentration. Similar to GSSG, the detected amount of glutathione sulfonic acid was found to be formed at least in part during the analytical process, but the degree could not be quantified.

New insights into the catalytic mechanism of Bombyx mori prostaglandin E synthase gained from structure–function analysis

Prostaglandin E synthase (PGES) catalyzes the isomerization of PGH2 to PGE2. We previously reported the identification and structural characterization of Bombyx mori PGES (bmPGES), which belongs to Sigma-class glutathione transferase. Here, we extend these studies by determining the structure of bmPGES in complex with glutathione sulfonic acid (GTS) at a resolution of 1.37Å using X-ray crystallography. GTS localized to the glutathione-binding site. We found that electron-sharing network of bmPGES includes Asn95, Asp96, and Arg98. Site-directed mutagenesis of these residues to create mutant forms of bmPGES mutants indicate that they contribute to catalytic activity. These results are, to our knowledge, the first to reveal the presence of an electron-sharing network in bmPGES.

Pre-Steady-State Kinetic Characterization of Thiolate Anion Formation in Human Leukotriene C4 Synthase

Human leukotriene C₄ synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C₄ (LTC₄). This molecule, together with its metabolites LTD₄ and LTE₄, induces inflammatory responses, particularly in asthma, and thus, the enzyme is an attractive drug target. During the catalytic cycle, glutathione (GSH) is activated by hLTC4S that forms a nucleophilic thiolate anion that will attack LTA₄, presumably according to an S(N)2 reaction to form LTC₄. We observed that GSH thiolate anion formation is rapid and occurs at all three monomers of the homotrimer and is concomitant with stoichiometric release of protons to the medium. The pK(a) (5.9) for enzyme-bound GSH thiol and the rate of thiolate formation were determined (k(obs) = 200 s⁻¹). Taking advantage of a strong competitive inhibitor, glutathionesulfonic acid, shown here by crystallography to bind in the same location as GSH, we determined the overall dissociation constant (K(d((GS) = 14.3 μM). The release of the thiolate was assessed using a GSH release experiment (1.3 s⁻¹). Taken together, these data establish that thiolate anion formation in hLTC4S is not the rate-limiting step for the overall reaction of LTC₄ production (k(cat) = 26 s⁻¹), and compared to the related microsomal glutathione transferase 1, which displays very slow GSH thiolate anion formation and one-third of the sites reactivity, hLTC4S has evolved a different catalytic mechanism.

Helix–helix interfaces and ligand binding

Helix–helix parallel interfaces can be characterized by certain combinations of amino acids, which repeatedly occur at core positions a and d (leucine zipper nomenclature) in homologous and nonhomologous proteins and influence interhelical angles. Applied for the prediction of interhelical angles in glutathione S-transferase, intracellular chloride channel and annexin molecules from various sources, correct results were achieved in 58 out of 62 proteins. Interhelical angles are found to correlate with the conformation of the glutathione S-transferase ligands glutathione, s-hexylglutathione, glutathione sulfonic acid, and glutathione-s-dinitrobenzene.

Glutathionylation of proteins by glutathione disulfide S-oxide

Aqueous solution of S-nitrosoglutathione (GSNO) underwent spontaneous chemical transformation that generated several glutathione derivatives including glutathione sulfonic acid (GSO3H), glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide, and glutathione disulfide. Surprisingly, GS(O)SG (also called glutathione thiosulfinate), which was not identified as a metabolite of GSNO previously, was one of the major products derived from GSNO. This compound was very reactive toward any thiol and the reaction product was a mixed disulfide. The rate of reaction of GS(O)SG with 5-mercapto-2-nitro-benzoate was nearly 20-fold faster than that of GSNO. The mechanism for the formation of GS(O)SG was believed to involve the sulfenic acid (GSOH) and thiosulfinamide (GS(O)NH2) intermediates; the former underwent self-condensation and the latter reacted with GSH to form GS(O)SG. Many reactive oxygen and nitrogen species were also capable of oxidizing GSH or GSSG to form GS(O)SG, which likely played a central role in integrating both the oxidative and nitrosative cellular responses through thionylation of thiols. Treatments of rat brain tissue slices with oxidants resulted in an enhanced thionylation of proteins with a concomitant increase in cellular level of GS(O)SG, suggesting that this compound might play a second messenger role for stimuli that produced a variety of oxidative species.

Study on glutathionesulfonic acid sodium salt as biodistribution promoter for thiopental sodium.

The effects of glutathione (GSH) and glutathionesulfonic acid sodium salt [N-(N-gamma-L-glutamyl-L-beta-sulfoalanyl)glycine sodium salt, GSO3Na], which is a minor metabolite of GSH, on the pharmacokinetics of thiopental sodium were investigated in rats. The concomitant use of GSO3Na with thiopental sodium significantly increased the tissue-to-plasma concentration ratio (Kp) of thiopental sodium 60 min after its administration in the heart, lung, brain, liver, kidney, and spleen, while GSH did not affect them. On the other hand, the Kp value of thiopental sodium 5 min after its administration with concomitant GSO3Na decreased significantly only in the spleen. Neither GSO3Na nor GSH changes the pharmacokinetic parameters of thiopental sodium. Significant change of the binding ratio of thiopental sodium to bovine serum albumin (BSA) was not observed by the addition of less than 5-fold GSO3Na. About 50% of thiopental sodium was bound to the brain, lung or liver, however, no significant change of this binding ratio was observed by the concomitant use of GSO3Na. The partition coefficient of thiopental sodium apparently increased by the concomitant use of GSO3Na but not by GSH. This phenomenon seemed to be concerned with a mechanism to increase the Kp values of thiopental sodium in the tissues. The increment in the drug distribution to tissues with concomitant GSO3Na observed in this study is useful information for the application of drug combinations as a biodistribution promoter.

Glutathione Oxidation by Hypochlorous Acid in Endothelial Cells Produces Glutathione Sulfonamide as a Major Product but Not Glutathione Disulfide

Treatment of cells with hypochlorous acid (HOCl) at sublethal doses causes a concentration-dependent loss in reduced glutathione (GSH) levels. We have investigated the products of the reaction of HOCl with GSH in human umbilical vein endothelial cells. Despite a complete loss of GSH, there were only very small increases in intracellular and extracellular glutathione disulfide and glutathione sulfonic acid after exposure to HOCl. (35)S labeling of the GSH pool showed only a minimal increase in protein-bound GSH, suggesting that S-thiolation was not a major contributor to HOCl-mediated loss of GSH in endothelial cells. Rather, the products of the reaction were mostly exported from cells and included a peak that co-eluted with the cyclic sulfonamide that is a product of the reaction of GSH with reagent HOCl. Evidence of this species in endothelial cell supernatants after HOCl treatment was also obtained using electrospray mass spectrometry. In conclusion, exposure to HOCl causes the irreversible loss of cellular GSH with the formation of novel products that are rapidly exported from the cell, and resynthesis of GSH will be required to restore levels. The loss of GSH would alter the redox state of the cell and compromise its defenses against further oxidative stress.

New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes

Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S -methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD +-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P) +-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn 2+ ion of FaDH and Arg 114, and by the hydrophobic contacts made with Tyr 92 and Met 140. Molecular models were also produced for the binding of BM1 and BM3 (glutathione-substituted) to GST I. In both cases the biomimetic dye forms multiple hydrophobic interactions with the enzyme through binding to a surface pocket. While the glutathioine moiety of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better purification results achieved with BM1.

Glutathiolation of Proteins by Glutathione DisulfideS-Oxide Derived from S-Nitrosoglutathione: MODIFICATIONS OF RAT BRAIN NEUROGRANIN/RC3 AND NEUROMODULIN/GAP-43

S-Nitrosoglutathione (GSNO) undergoes spontaneous degradation that generates several nitrogen-containing compounds and oxidized glutathione derivatives. We identified glutathione sulfonic acid, glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide, and GSSG as the major decomposition products of GSNO. Each of these compounds and GSNO were tested for their efficacies to modify rat brain neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm). Among them, GS(O)SG was found to be the most potent in causing glutathiolation of both proteins; four glutathiones were incorporated into the four Cys residues of Ng, and two were incorporated into the two Cys residues of Nm. Ng and Nm are two in vivo substrates of protein kinase C; their phosphorylations by protein kinase C attenuate the binding affinities of both proteins for calmodulin. When compared with their respective unmodified forms, the glutathiolated Ng was a poorer substrate and glutathiolated Nm a better substrate for protein kinase C. Glutathiolation of these two proteins caused no change in their binding affinities for calmodulin. Treatment of [(35)S]cysteine-labeled rat brain slices with xanthine/xanthine oxidase or a combination of xanthine/xanthine oxidase with sodium nitroprusside resulted in an increase in cellular level of GS(O)SG. These treatments, as well as those by other oxidants, all resulted in an increase in thiolation of proteins; among them, thiolation of Ng was positively identified by immunoprecipitation. These results show that GS(O)SG is one of the most potent glutathiolating agents generated upon oxidative stress.

The Catalytic Tyr-9 of Glutathione S-Transferase A1-1 Controls the Dynamics of the C terminus

The glutathione S-transferase enzymes (GSTs) have a tyrosine or serine residue at their active site that hydrogen bonds to and stabilizes the thiolate anion of glutathione, GS(-). The importance of this hydrogen bond is obvious, in light of the enhanced nucleophilicity of GS(-) versus the protonated thiol. Several A-class GSTs contain a C-terminal segment that undergoes a ligand-dependent local folding reaction. Here, we demonstrate the effects of the Y9F substitution on binding affinity for glutathione conjugates and on rates of the order-disorder transition of the C terminus in rat GST A1-1. The equilibrium binding affinity of the glutathione conjugate, GS-NBD (NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole), was decreased from 4.09 microm to 0.641 microm upon substitution of Tyr-9 with Phe. This result was supported by isothermal titration calorimetry, with K(d) values of 1.51 microm and 0.391 microm for wild type and Y9F, respectively. The increase in binding affinity for the mutant is associated with dramatic decreases in rates for the C-terminal order-disorder transition, based on a stopped-flow kinetic analysis. The same effects were observed, qualitatively, for a second GSH conjugate, GS-ethacrynic acid. Apparently, the phenolic hydroxyl group of Tyr-9 is critical for orchestrating C-terminal dynamics and efficient product release, in addition to its role in lowering the pK(a) of GSH.

Glutathione oxidation in real time by thermospray liquid chromatography–mass spectrometry

The oxidation and reduction of glutathione and oxidized glutathione were studied in real time by liquid chromatography–mass spectrometry during exposure to hydrogen peroxide and mercaptoethanol. By mass spectrometry mixed disulfides and both reversible and irreversible oxidations of sulfur to higher states (sulfinic and sulfonic acids) were directly observed during exposure to hydrogen peroxide. The irreversible oxidation of glutathione to glutathione sulfonic acid could be detected after 30 min exposure of glutathione to 40 m M H 2O 2 at 20°C. A peak consistent with glutathione-sulfinic acid was transiently present, suggesting this compound behaved as an oxygen consuming antioxidant. Liquid chromatography–mass spectrometry appears to be an excellent method to study oxidation and reductions of sulfur containing peptides and amino acids.

Influence of Glutathione Oxidation and pH on Thermal Formation of Maillard-Type Volatile Compounds

The Maillard-type volatile compounds obtained from the reaction of glutathione and glucose were mainly furans, carbonyl compounds, and sulfur-containing compounds including thiophenes, thiazoles, and cyclic polysulfides. Both pH 8.0 and 6.0 were favorable conditions for sulfur-containing compound formation, whereas acidic conditions were favorable for furan and derivative formation. The reaction between glutathionesulfonic acid, an oxidized form of glutathione, and glucose primarily produced furans, carbonyl compounds, pyrazines, and pyrroles. Furans dominated the products obtained from the reactions at pH 6.0 and 8.0. Pyrazines increased as the reaction pH increased. The disappearance of sulfur-containing compounds in the products of glutathionesulfonic acid and glucose reaction systems indicated glutathionesulfonic acid could not provide hydrogen sulfide for the reaction. Keywords: Glutathione; glutathionesulfonic acid; sulfur-containing; oxidation; pH

Influence of Protein-Glutathione Mixed Disulfide on the Chaperone-like Function of α-Crystallin

In an earlier report we showed that incubation of alpha-crystallin with oxidized glutathione results in significant loss of its chaperone-like activity. In the present study, we determined the effect of protein-glutathione mixed disulfides (PSSG), formed at Cys-131 in bovine alphaA-crystallin, and Cys-131 and Cys-142 in human alphaA-crystallin, on the function of alpha-crystallin as a molecular chaperone. After incubation of calf and young human alphaL-crystallin fractions with oxidized glutathione, levels of PSSG were determined by performic acid oxidation of the mixed disulfides followed by reversed-phase high pressure liquid chromatography separation of phenylisothiocyanate-derivatized glutathione sulfonic acid. Levels of PSSG increased from 0.01 to 0.14 nmol/nmol (20 kDa) in bovine alphaL-crystallin and from 0.022 to 0.25 nmol/nmol in human alphaL-crystallin. The presence of glutathione adducts at Cys-131 and Cys-142 were confirmed by mass spectral analysis. The chaperone-like activity was determined by the heat denaturation assay using betaL-crystallin as the target protein. To examine the reversibility of the effect of mixed disulfides on chaperone activity, studies were done before and after reduction with the glutathione reductase system. Increased levels of PSSG resulted in lower chaperone activities. Treatment with the glutathione reductase system led to 80% reduction in PSSG levels with a concomitant recovery of the chaperone activity. These results suggest that cysteine(s) in the alphaA-crystallin subunit play an important role in the function of alpha-crystallin as a molecular chaperone.

High-Performance Liquid Chromatography Method for the Determination of Protein-Glutathione Mixed Disulfide

We have developed an efficient and sensitive method for the quantification of picomole quantities of protein-glutathione and protein-cysteine mixed disulfides in ocular lens. Performic acid oxidation releases glutathione and cysteine from the protein-mixed disulfides as glutathione sulfonic acid and cysteic acid, respectively. The sulfonic acids are derivatized with phenylisothiocyanate, and the phenylthiocarbamyl derivatives are separated by HPLC using a reversed-phase ODS column, equilibrated with 0.14 M sodium acetate, containing 0.1% triethylamine and 6% acetonitrile.

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95.

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.

Analysis of strongly acidic amino acids by the conventional amino acid analyzer: Application to determination of protein-bound cysteine and glutathione

A rapid analysis method of strongly acidic amino acids and related compounds by a simple modification of an existing amino acid analyzer is presented. In this method, an anion-exchanger column (2.6 × 150 mm) packed with Hitachi 3013-N resin was developed with 0.2 m citric acid. Complete separation of phosphothreonine, phosphoserine, phosphotyrosine, cysteic acid, homocysteic acid, and glutathionesulfonic acid was achieved within 35 min, with no regeneration of the column being required. Tyrosine- O-sulfate was analyzed by the same column using 2 m sodium acetate buffer, pH 5.5. Performic acid oxidation of a variety of proteins and direct analysis of the products by this system successfully detected cysteine, homocysteine, and/or glutathione bound to proteins through disulfide bonds. This suggests the potential use of the method for analysis of the states of protein thiol groups, especially those of clinically significant mutant proteins where mutation of arginine to cysteine is rather frequently recognized.

Quantitation of lens protein mixed disulfides by ion-exchange chromatography

A sensitive and high resolution ion-exchange chromatographic method was employed to analyse lens protein mixed disulfides. The mixed disulfides were analysed and quantified as glutathione sulfonic acid and cysteic acid after the lens proteins were subjected to performic acid oxidation. The oxidized product was applied on a 0·9×15 cm column packed with BioRad Aminex A-28 resin (9 μm beads). The column was maintained at 55°C and eluted with 0·3 m sodium acetate buffer, pH 5·1 at 60 ml hr −1 for 40 min before switching to 0·5 m of the same buffer. The eluate was subsequently reacted with ninhydrin and monitored for absorbance at 570 nm. The glutathione sulfonic acid emerged at 69 min, cysteic acid at 35 min, and both were clearly resolved from neighboring peaks and could be detected at < nmol level. In the lens, both glutathione and cysteine protein mixed disulfides were only found in the TCA-insoluble protein fraction. Normal rat lens contained 0·35 nmol per lens of protein-bound glutathione but 10-fold this amount of protein-bound cysteine. The hyperglycemic condition had little effect on the protein thiol mixed disulfide level except that at a more advanced diabetic condition, equal depletion of cysteine and glutathione mixed disulfides were found. On the other hand, oxidative conditions induced a marked elevation of glutathione mixed disulfide but not of the cysteine mixed disulfide.

Glutathionesulfonic Acid, the Predominant Product of X-Radiolysis of Air-Saturated Solutions of Oxidized Glutathione

Glutathionesulfonic acid is identified as the sole product of exposure of dilute, air-saturated aqueous solutions of the hexapeptide disulfide oxidized glutathione to doses of X-rays sufficient to destroy up to 80% of the starting material. The radiation-chemical yields are similar to those of cysteic acid from cystine and taurine from cystamine under similar conditions and exhibit concentration dependence similar to that shown by these simpler disulfides. Qualitative interpretation of the results is consistent with the proposal that the initiating step is electron abstraction from the disulfide giving a cation-radical.