Glutathiyl Radicals Research Articles

ChemInform Abstract: Repair of Protein Radicals by Antioxidants

AbstractReview: 119 refs.

Repair of Protein Radicals by Antioxidants

AbstractIn vivo, proteins are the main targets for radicals and other reactive species. Their reactions result in formation of amino acid radicals on the protein surface that often yield tryptophan and tyrosyl radicals or, in the presence of O2, protein peroxyl radicals and hydroperoxides. All these species may propagate damage to biomolecules. Low molecular weight antioxidants, such as ascorbate, urate, and glutathione, are part of the defense system and function by repairing damaged proteins. We briefly review the existing knowledge about protein and amino acid radicals and their repair by antioxidants, including results of our investigations. The main question addressed is whether the antioxidants ascorbate, urate, and glutathione are able to repair amino acid radicals in model compounds and in proteins in vitro by pulse radiolysis. We show that ascorbate and urate repair tryptophan and tyrosyl radicals efficiently and inhibit proton‐coupled electron transfer from tyrosine residues to tryptophan radicals in a number of proteins. In contrast, repair by glutathione is much slower. Ascorbate also rapidly reduces the peroxyl radicals of the N‐acetylamide derivatives of glycine, alanine, and proline, whereas glutathione reduces peroxyl radicals in lysozyme. In vivo urate, ascorbate, and glutathione may prevent biological damage or, at least, reduce its rate, because they: (a) repair tryptophan and tyrosyl radicals in proteins and (b) reduce protein peroxyl radicals to the corresponding protein hydroperoxides. Most likely, in vivo, ascorbate and glutathione do not inhibit the reaction of C‐centered amino acid radicals with O2. Glutathione is less efficient that urate and ascorbate in repairing protein radicals; furthermore, the resulting glutathiyl radical is harmful. Ascorbate may be the more important repair agent in cells and tissues characterized by high ascorbate concentrations, such as the lens and brain; urate may be mainly responsible for repair in tissue compartments with higher urate concentrations, such as in plasma and saliva.

Glutathiyl radical as an intermediate in glutathione nitrosation

Nitrosation of thiols is thought to be mediated by dinitrogen trioxide (N2O3) or by nitrogen dioxide radical (NO2). A kinetic study of glutathione (GSH) nitrosation by NO donors in aerated buffered solutions was undertaken. S-nitrosoglutathione (GSNO) formation was assessed spectrophotometrically and by chemiluminescence. The results suggest an increase in the rate of GSNO formation with an increase in GSH with a half-maximum constant EC50 that depends on NO concentration. Our observed increase in EC50 with NO concentration suggests a significant contribution of NO2-mediated nitrosation with the glutathiyl radical as an intermediate in the production of GSNO.

Experimental results and theoretical validation for the antioxidant mechanism of bilirubin

The present study demonstrates that hydroxyl, 1-hydroxy ethyl and glutathiyl radicals abstract hydrogen atom from bilirubin and forms a carbon-centered radical. The bimolecular rate constant for the reaction of ·OH, 1-hydroxy ethyl and glutathiyl radicals with bilirubin are 1.0 x 10 10 , 2.0 × 10 8 and 6.0 x 10 8 dm 3 mol -1 s -1 , respectively. The transient absorption spectra obtained in these reactions have two peaks at 350 nm and 540 nm with bleaching in absorption at 450 nm. Both the absorption peaks form and decay with same rate constants showing that they are due to the same species. The bilirubin radical thus produced, decays with first order rate constant of 1.13 x 10 4 s -1 . In presence of oxygen the decay becomes faster confirming it to be a carbon-centered radical. Linoleic acid peroxyl radical also reacts with bilirubin via hydrogen abstraction. In the case of NO 2 · radical reaction, bilirubin produces absorption peak at 590 nm assigned to be the radical cation formed via single electron transfer from bilirubin. The bimolecular rate constant for this reaction is 2.0 x 10 9 dm 3 mol -1 s -1 . Corrected absorption spectra have been shown in few cases of these oxidation reactions. These results show that the oxidation of bilirubin can occur in two ways, either via hydrogen atom transfer or via single electron transfer depending on the nature of the oxidizing radical. Ab initio molecular orbital calculations have been performed to investigate the structure and energetics of radicals and radical cation produced by removal of hydrogen and electron, respectively from bilirubin molecule. It has been found that hydrogen abstraction from central methylenic group is most probable whereas pyrrole radical cation is most stable.

Electron Paramagnetic Resonance Spin Trapping of Glutathiyl Radicals by PBN in the Presence of Cyclodextrins and by PBN Attached to β-Cyclodextrin

The reaction of the glutathiyl radical (GS*) with a widely used spin trap N- tert-butyl-alpha-phenylnitrone (PBN) has been studied in the presence of various methylated beta-cyclodextrins and with PBN covalently bound to dimethylated beta-cyclodextrin (PBN-DIMEB) and permethylated beta-cyclodextrin (PBN-TRIMEB). Scavenging rate constants for GS* by PBN were obtained in the presence of randomly methylated cyclodextrin (RAMEB) and PBN-TRIMEB and found to be close to the rate constant previously measured for PBN. RAMEB and 2,6-di- O-Me-beta-cyclodextrin (DIMEB) were found to be the most efficient in the increasing PBN/GS* lifetime, by a factor of 5.5 for RAMEB and 6.8 for DIMEB compared with the lifetime of PBN/GS*. It is concluded that the presence or "attachment of" beta-cyclodextrins does not influence the scavenging rate constant of GS* but it does lead to stabilization of the spin adducts formed.

Mechanistic Studies of the Reactions of Nitrone Spin Trap PBN with Glutathiyl Radical

We performed mechanistic studies of the reaction of PBN with the physiologically relevant glutathiyl radical, GS*, formed upon oxidation of the intracellular antioxidant, glutathione, GSH. The scavenging rate constant of GS* by PBN has been measured directly by laser flash photolysis and indirectly by competitive EPR of the spin adduct of PBN and another spin trap, DMPO (5,5-dimethyl-1-pyrroline N-oxide), and was found to be 6.7 x 107 M(-1) s(-1). Reverse decomposition of the paramagnetic PBN-glutathiyl radical adduct to the nitrone and thiyl radical was observed for the first time. The rate constant for the reaction of the monomolecular decomposition of the radical adduct was found to be 1.7 s(-1). Diamagnetic, EPR-invisible products of PBN adduct degradation were studied by 1H NMR and 19F NMR using newly synthesized fluorine-substituted PBN.

Detection, Characterization, and Decay Kinetics of ROS and Thiyl Adducts of Mito-DEPMPO Spin Trap

We report here the detection and characterization of spin adducts formed from the trapping of reactive oxygen species (superoxide and hydroxyl radicals) and glutathiyl and carbon-centered radicals by a newly synthesized nitrone, Mito-DEPMPO. This is a cationic nitrone spin trap with a triphenyl phosphonium cation conjugated to the DEPMPO analogue. The Mito-DEPMPO-OOH adduct, formed from the trapping of superoxide by Mito-DEPMPO, was enzymatically generated using xanthine/xanthine oxidase and neuronal nitric oxide synthase, and chemically generated by KO2 in 18-crown-6. The Mito-DEPMPO-OOH adduct exhibits an eight-line EPR spectrum with partial asymmetry arising from the alternate line-width effect. The half-life of the Mito-DEPMPO-OOH adduct is 2-2.5-times greater than that of the DEPMPO-OOH. The Mito-DEPMPO-SG adduct, formed from the trapping of glutathiyl radicals by Mito-DEPMPO, is 3-times more persistent than the analogue DEPMPO-SG adduct. In this study, we describe the EPR characterization of spin adducts formed from Mito-DEPMPO. The EPR parameters of Mito-DEPMPO adducts are distinctly different and highly characteristic. The detection of superoxide from an intact mitochondrion was feasible with Mito-DEPMPO but not with DEPMPO. We conclude that Mito-DEPMPO nitrone and its analogues are more effective than most nitrone spin traps for trapping superoxide, hydroxyl, and thiyl radicals formed in biological systems, including mitochondria.

Nitroxidative, Nitrosative, and Nitrative Stress: Kinetic Predictions of Reactive Nitrogen Species Chemistry Under Biological Conditions

A freely available Windows-based program, RNSim1A, is utilized to predict metal-independent reactive nitrogen species (RNS) chemistry (oxidation, nitrosation, and nitration) under simulated biological conditions and make the following specific predictions. (1) The peak in oxidative reactions that occurs in vitro with 1:1 fluxes of (*)NO and O(2)(*)(-) does not occur under biological conditions. (2) By far, the quantitatively dominant (92-99.6%) process in vivo is oxidation, compared to nitrosation and nitration. (3) Only five of the many possible RNS reactions involving thiol (glutathione, GSH) and tyrosine are quantitatively important biologically. (4) Under inflammatory conditions, approximately 1% of O(2)(*)(-) reacts with (*)NO to produce ONOO(-), with the remainder reacting with SOD. (5) The dominant reaction of tyrosyl radical is a radical swap with GSH, producing the glutathiyl radical and regenerating tyrosine. (6) Nitrosothiol is formed virtually exclusively via radical recombination (RS(*) + (*)NO) as opposed to reaction with nitrous anhydride (N(2)O(3)). (7) Nitrosothiol is an intermediate, not an endproduct, and responds dynamically to changes in the immediate chemical environment. (8) The formation of a nitroso group on a particular thiol can be considered a marker of increased reactivity of that thiol, and it is likely that other modifications of that thiol (oxidation, glutathiolation) are more abundant than nitrosation and may be the functionally significant modification. (9) Specific chemical mechanisms are proposed for posttranslational protein modification via nitrosation, nitration, glutathiolation, and also dithiol/disulfide exchange, with important roles for the thiolate anion and O(2) (suggesting possible mechanisms for O(2) sensing) and variable degrees of exposure of cysteine thiol and tyrosine phenolate. (10) Patterns of reactivity are similar for low (20 nM) and high (500 nM) steady-state levels of NO. (11) The dominant reactions are those involving reactants at the highest concentrations (CO(2), thiol, O(2)). Because of the dominance of oxidative processes caused by RNS, the term nitroxidative stress is proposed, emphasizing the oxidative (as opposed to nitrosative or nitrative) stress that dominates RNS actions under biological conditions.

The radioprotection and antioxidant properties of dehydrogingerdione

The reactions of hydroxyl and glutathiyl radicals with dehydrogingerdione (1) have been studied using biochemical assays and the pulse radiolysis technique in order to understand its radioprotective and antioxidant action. It was found to inhibit the Fenton-mediated 2-deoxyribose oxidation, as well as γ-ray-induced plasmid DNA strand breaks in a concentration-dependent fashion. The pulse radiolysis studies revealed its high reactivity with the hydroxyl and glutathiyl radicals. With the hydroxyl radicals, it initially formed three species, an adduct, a phenoxyl and a methylenic radical. The bimolecular rate constants for the formation of these species were the same (approx. 6.8 × 109 dm3/mol per s). The initially formed adduct also produced the phenoxyl radical at a later stage. In the case of glutathiyl-radical-induced oxidation, an adduct at the α–β double bond was formed as the sole species. Its identity as a carbon-centered radical was inferred from the result of the oxygen effect on the decay of the adduct radical. At a later stage, the adduct radical also produced the phenoxyl radical through an intramolecular radical transformation. A suitable mechanism for the oxidation reactions of 1 has been proposed.

Antioxidant properties of Plumbago zeylanica, an Indian medicinal plant and its active ingredient, plumbagin

Plumbago zeylanica (known as 'Chitrak') is a useful Indian medicinal plant. The root of the plant and its constituents are credited with potential therapeutic properties including anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective properties. To examine possible mechanisms of action of P. zeylanica (Chitrak), in relation to its reported beneficial properties, antioxidant effects of the aqueous/alcoholic extracts of root, corresponding to medicinal preparations, and the active ingredient, plumbagin, were studied. Methods used included: ferric reducing/antioxidant power (FRAP), radical scavenging of 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS), lipid peroxidation in rat liver mitochondria induced by different agents, and estimating phenolic and flavonoid content. In FRAP/DPPH assays, boiled ethanolic extracts were the most effective, while in the ABTS assay boiled aqueous extracts were the most efficient. These extracts also significantly inhibited lipid peroxidation induced by cumene hydroperoxide, ascorbate-Fe2+ and peroxynitrite and contained high amounts of polyphenols and flavonoids. To examine the mechanisms of action in detail, antioxidant and pulse radiolysis studies with plumbagin were conducted. The hydroxyl (•OH), alkyl peroxyl (CCl3OO•), linoleic acid peroxyl (LOO•), and glutathiyl (GS•) radicals generate a phenoxyl radical upon reaction with plumbagin. The bimolecular rate constants were: •OH, 2.03 x 109 dm3mol–1s–1; CCl3OO•, 1.1 x 109 dm3mol–1s–1; LOO•, 6.7 x 107 dm3mol–1s–1; and GS•, 8.8 x 108 dm3mol–1s–1. In conclusion, our studies reveal that extracts of P. zeylanica and its active ingredient plumbagin have significant antioxidant abilities that may possibly explain some of the reported therapeutic effects.

Reversible Reactions of Thiols and Thiyl Radicals with Nitrone Spin Traps

The reactions of the reversible addition of thiols and thiyl radicals to the nitrone spin traps DMPO (5,5-dimethyl-1-pyrroline N-oxide) and DEPMPO (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide) are described. Addition of the thiols to the double CN bond of the nitrones results in the formation of the corresponding hydroxylamines, measured using 31P NMR and the phosphorus-containing trap DEPMPO. Subsequent mild oxidation of these hydroxylamines into the paramagnetic adducts may interfere with genuine spin trapping of thiyl radicals representing the Forrester−Hepburn mechanism. The reverse decomposition of hydroxylamines to the parent nitrone and thiol and of paramagnetic adducts to the nitrone and thiyl radical were observed for the first time. The recycling of reduced thiols from thiyl radicals by nitrones may comprise the mechanism of their effective antioxidant activity, in vivo. The release of thiyl radicals upon the breakdown of the paramagnetic adduct may significantly affect not only the quantitative analysis of the spin trapping data but even the conclusions regarding the origin of short-lived radical intermediates. The equilibrium constant for the reactions of the formation of the product of DEPMPO with S-centered nucleophiles decreases in the series: sulfite > thioglycolic acid > cysteine > glutathione. The rate constants for the reaction of the monomolecular decomposition of the radical adducts back to the nitrone and glutathiyl radical were found to be equal to 0.3 ± 0.1 s-1 and 0.02 s-1 for DMPO/GS• and DEPMPO/GS• radical adducts, correspondingly.

Protein Radical Formation during Lactoperoxidase-mediated Oxidation of the Suicide Substrate Glutathione: IMMUNOCHEMICAL DETECTION OF A LACTOPEROXIDASE RADICAL-DERIVED 5,5-DIMETHYL-1-PYRROLINE N-OXIDE NITRONE ADDUCT

A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H(2)O(2). The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.

EPR detection of glutathiyl and hemoglobin-cysteinyl radicals during the interaction of peroxynitrite with human erythrocytes.

Peroxynitrite, which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of human diseases. An initial controversy about the possibility of free radical production from peroxynitrite in test tubes has been resolved, and presently it is important to establish whether peroxynitrite produces radicals in cells. Here we employed the EPR spin trapping methodology with 5,5-dimethylpyrroline N-oxide (DMPO) to study the interaction of peroxynitrite with human erythrocytes. The results confirmed previous findings in demonstrating that oxyhemoglobin is the main target of peroxynitrite in erythrocytes. As we first show here, the produced ferryl-hemoglobin oxidizes its own amino acids and, most probably, amino acids from other hemoglobin monomers to produce hemoglobin-tyrosyl and hemoglobin-cysteinyl radicals. In parallel, ferryl-hemoglobin also oxidizes intracellular glutathione to produce the glutathiyl radical. The EPR spectrum of both DMPO/(*)cysteinyl-hemoglobin (a(beta)(H) = 15.4 G) and DMPO/(*)tyrosyl-hemoglobin (a(beta)(H) = 8.8 G) radical adducts was characterized. It is proposed that erythrocytes can be efficient peroxynitrite scavengers in vivo through the coupled action of oxyhemoglobin and glutathione. Overall, the results indicate that, through the intermediacy of carbon dioxide and/or hemoproteins, oxidation of glutathione to the glutathiyl radical is likely to be an important consequence of peroxynitrite production in vivo.

Synthesis and biochemical applications of a solid cyclic nitrone spin trap: a relatively superior trap for detecting superoxide anions and glutathiyl radicals

A novel cyclic nitrone spin trap, 5- tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) as a pure white solid has been synthesized for the first time. BMPO offers several advantages over the existing spin traps in the detection and characterization of thiyl radicals, hydroxyl radicals, and superoxide anions in biological systems. The corresponding BMPO adducts exhibit distinct and characteristic electron spin resonance (ESR) spectral patterns. Unlike the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-derived superoxide adduct, the BMPO superoxide adduct does not non-enzymatically decompose to the BMPO hydroxyl adduct. This feature is clearly perceived as a definite advantage of BMPO in its biological applications. In addition, the ESR spectrum of the BMPO glutathionyl adduct (BMPO/ SG) does not fully overlap with the spectrum of its hydroxyl adduct. This spectral feature is again distinctly different from that of DMPO because the ESR spectral lines of DMPO glutathionyl and hydroxyl radical adducts largely overlap. Finally, the ESR spectra of BMPO-derived adducts exhibit a much higher signal-to-noise ratio in biological systems. These favorable chemical and spectroscopic features make BMPO ideal for the detection of superoxide anions, hydroxyl and thiyl radicals in biochemical oxidation and reduction.

Inhibition of peroxynitrite-induced nitration of tyrosine by glutathione in the presence of carbon dioxide through both radical repair and peroxynitrate formation.

Peroxynitrite (ONOO-/ONOOH) is assumed to react preferentially with carbon dioxide in vivo to produce nitrogen dioxide (NO2*) and trioxocarbonate(1-) (CO3*-) radicals. We have studied the mechanism by which glutathione (GSH) inhibits the NO2*/CO3*--mediated formation of 3-nitrotyrosine. We found that even low concentrations of GSH strongly inhibit peroxynitrite-dependent tyrosine consumption (IC50 = 660 microM) as well as 3-nitrotyrosine formation (IC50) = 265 microM). From the determination of the level of oxygen produced or consumed under various initial conditions, it is inferred that GSH inhibits peroxynitrite-induced tyrosine consumption by re-reducing (repairing) the intermediate tyrosyl radicals. An additional protective pathway is mediated by the glutathiyl radical (GS*) through reduction of dioxygen to superoxide (O2*-) and reaction with NO2* to form peroxynitrate (O2NOOH/O2NOO-), which is largely unreactive towards tyrosine. Thus, GSH is highly effective in protecting tyrosine against an attack by peroxynitrite in the presence of CO2. Consequently, formation of 3-nitrotyrosine by freely diffusing NO2* radicals is highly unlikely at physiological levels of GSH.

Photochemical sensitization by azathioprine and its metabolites. Part 3. A direct EPR and spin-trapping study of light-induced free radicals from 6-mercaptopurine and its oxidation products.

Sunlight has been implicated in the high incidence of skin cancer found in patients receiving 6-mercaptopurine (PSH) in the form of its pro-drug azathioprine. In this study we have used EPR spectroscopy in conjunction with the spin-trapping technique to determine whether PSH and its metabolic or photochemical oxidation products generate highly reactive free radicals upon UV irradiation. When an aqueous anaerobic solution (pH 5 or 9) of PSH (pKa = 7.7) and either 2-methyl-2-nitrosopropane (MNP) or nitromethane (NM) were irradiated (lambda > 300 nm) with a Xe arc lamp, the corresponding purine-6-thiyl (PS.) radical adduct and the reduced form of the spin trap (MNP/H. or CH3NO2.-) were observed. However, no radical adducts were detected when PSH and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were irradiated (lambda = 320 nm) in oxygen-free buffer. These findings suggest that PSH does not photoionize but that instead MNP and NM are reduced by direct electron transfer from excited state PSH, 1.3(PSH)*. In aerobic solution, oxygen can act as an electron acceptor and the O2.- and PS. radicals are formed and trapped by DMPO. 6-Mercaptopurine did photoionize when irradiated with a Nd:YAG laser at 355 nm as evidenced by the appearance of the DMPO/H.(eq- + H+) adduct, which decreased in intensity in the presence of N2O. 1.3(6-Mercaptopurine)* oxidized ascorbate, formate and reduced glutathione to the corresponding ascorbyl, CO2.- or glutathiyl radicals. The photochemical behavior of 6-thioxanthine and 6-thiouric acid was similar to PSH. However, the excited states of these metabolic oxidation products exhibited stronger reducing properties than 1.3(PSH)*.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiol and Mn(2+)-mediated oxidation of veratryl alcohol by horseradish peroxidase

Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).