An ESR investigation of the reactions of glutathione, cysteine and penicillamine thiyl radicals: competitive formation of RSO., R., RSSR-., and RSS(.)

The reactions of the cysteine, glutathione and penicillamine thiyl radicals with oxygen and their parent thiols in frozen aqueous solutions have been elucidated through electron spin resonance spectroscopy. The major sulfur radicals observed are: (1) thiyl radicals, RS.; (2) disulfide radical anions. RSSR-.; (3) perthiyl radicals, RSS. and upon introduction of oxygen; (4) sulfinyl radicals, RSO., where R represents the remainder of the cysteine, glutathione or penicillamine moiety. The radical product observed depends on the pH, concentration of thiol, and presence or absence of molecular oxygen. We find that the sulfinyl radical is a ubiquitous intermediate in the free radical chemistry of these important biological compounds, and also show that peroxyl radical attack on thiols may lead to sulfinyl radicals. We elaborate the observed reaction sequences that lead to sulfinyl radicals, and, using 17O isotopic substitution studies, demonstrate that the oxygen atom in sulfinyl radicals originates from dissolved molecular oxygen. In addition, the glutathione thiyl radical is found to abstract hydrogen from the alpha-carbon position on the cysteine residue of glutathione to form a carbon-centered radical.     

An ESR study of radical kinetics in L-alpha-amino-n-butyric acid hydrochloride containing L-cysteine hydrochloride

On annealing at temperatures near 100 degrees C, carbon-centered radicals migrate to sulfur-centered radicals in X-irradiated crystals of L-alpha-amino-n-butyric acid hydrochloride, CH3CH2CH(NH3-Cl)COOH, containing L-cysteine hydrochloride, SHCH2CH(NH3Cl)COOH. Samples containing 0, 0.5, 1.0, and 1.5% L-cysteine hydrochloride were studied. When no cysteine is present, the carbon-centered radical formed by X irradiation, CH3CH2CHOOH, decays according to a second-order diffusion-controlled rate equation. In samples containing cysteine, the same carbon-centered radicals are formed, but on annealing, they migrate to cysteine, where a perithiyl radical, RSS, is formed. The transfer of carbon-centered radicals to perthiyl radicals follows a pseudo first-order rate equation with an activation energy of 1.15 eV. A decrease in the initial concentration of the carbon-centered radicals or an increase in the initial concentration of cysteine results in an increase in the transfer efficiency. The rate of growth of the perthiyl radical depends on both the initial concentration of cysteine and the initial concentration of carbon-centered radicals. The pseudo first-order rate constant increases when either the initial carbon-centered radical concentration increases or the initial cysteine concentration increases. The mechanism by which radicals move from one lattice site to another in the crystalline material is most likely hydrogen abstraction from a neighboring molecule.     

Study on the mechanism of action of artemether against schistosomes: the identification of cysteine adducts of both carbon-centred free radicals derived from artemether

Reaction of the antimalarial and anti-schistosome drug artemether (1) and catalytic amount of ferrous ion in the presence of excess cysteine gave two adducts of cysteine and previous postulated primary and secondary carbon-centred free radicals besides their other rearrangement products. This piece of further evidence for the presence of carbon-centred radicals, especially the secondary carbon-centred free radical for the first time by the isolation of its coupling adduct, will be help to understand the mechanism of action of artemether and other qinghaosu derivatives against parasites.     

Synergistic inhibition of oxidation in dispersed phosphatidylcholine liposomes by a combination of vitamin E and cysteine

Oxidations of soybean phosphatidylcholine liposomes in an aqueous dispersion initiated by free radicals generated initially either in the aqueous phase or in the lipid phase were efficiently suppressed by vitamin E in the membranes. Vitamin E was consumed linearly with time and, when the inhibition period was over the oxidation proceeded rapidly at a rate similar to that in the absence of vitamin E. L-Cysteine was also effective by itself in scavenging radicals in the aqueous region, but it was consumed more rapidly than vitamin E. On the other hand, cysteine could not scavenge the radicals efficiently in a lipid region. Nevertheless, when vitamin E was incorporated into liposomes, the addition of cysteine in the aqueous phase prolonged the inhibition period and it reduced the rate of decay of vitamin E markedly even when the radicals were generated initially in the lipid bilayer. Furthermore, it was found by an electron spin resonance study that chromanoxyl radical disappeared quite rapidly when it was mixed with cysteine and that the spin adduct of cysteine radical was observed in the presence of alpha-(4-pyridyl-N-oxide)-N-tert-butyl nitrone. It was concluded that L-cysteine located in an aqueous region could regenerate vitamin E by reacting with vitamin E radical formed in a lipid region and show a synergistic antioxidant effect, although its efficiency of vitamin E regeneration was lower than that by vitamin C.     

Kinetics of polymerization of hemoglobin S modified by thiol reagents and by oxidation

The effects of four thiol reagents on the kinetics of polymerization of hemoglobin S have been studied in high phosphate buffer (1.8 M), in the presence (3 mM) or absence of sodium dithionite, depending on the reduction of mixed disulfides of Hb in the presence of this reducing agent. The effect of oxidized forms (methemoglobin) of HbS on the kinetics of aggregation of deoxyHbS was also studied because of the presence of 33% metHbS when HbS was modified by 4-aminophenyl disulfide. In the presence of sodium dithionite, the delay times prior to polymerization of deoxyHbS modified by N-ethylmaleimide, iodoacetamide and 4-aminophenyl disulfide were, respectively, 1.5-, 1.35- and 1.15-times longer than that of native deoxyHbS. The results indicate that the radicals bound to the cysteine beta 93 residue inhibit the contacts in the polymer formation to various extents but do not modify the size of the nuclei.     

Study of activated oxygen production by some thiols using chemiluminescence.

2-3-dimercapto-1-propane sulfonic acid, D-penicillamine and meso-dimercapto succinic acid, drugs widely applied as antidota against metal poisoning, and cysteine and glutathione were studied with respect to their ability to generate and to scavenge superoxide anion radical. Superoxide production and scavenging were tested by means of luminol-dependent chemiluminescence. In presence of 1 mumol/l ADP-Fe3+ only cysteine and meso-dimercapto succinic acid induced chemiluminescence which could be inhibited by superoxide dismutase. 2,3-dimercapto-1-propane sulfonic acid, D-penicillamine and glutathione acted as O2- scavengers. These thiols inhibited O2(-)-dependent lipid peroxidation thus acting as antioxidants, whereas cysteine and meso-dimercapto succinic acid accelerated peroxidation. It is suggested that the toxic side effects of thiols may be due to their ability to generate or to scavenge free radicals.     

Effects of sulphydryl compounds on the radiation damage in biologically active DNA

The effect of sulphydryl compounds on the induction of alkali-labile sites and on the contribution of such sites to the inactivation of single-stranded phi X174 DNA was studied. Cysteamine is capable of reacting with DNA radicals, thereby modifying the radiation damage in such a way that the induction of immediate and latent breaks is reduced. This depends on the pH of the solution. With cysteine only, a pH dependent protection, against lethal alkali-labile potential breaks could be observed. The damage other than breaks is not influenced by the presence of sulphydryl compounds.     

Free radical metabolites of L-cysteine oxidation

The oxidation of L-cysteine by horseradish peroxidase in the presence of oxygen forms a thiyl free radical as demonstrated with the spin-trapping ESR technique. Reactions of this thiyl free radical result in oxygen consumption, which is inhibited by the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. Cysteine sulfinic acid, a cysteine metabolite, is a poorer substrate for horseradish peroxidase than cysteine and is oxidized to form both sulfur-centered and carbon-centered free radicals.     

Pro-oxidant activity of apocynin radical

Apocynin has been widely used as an NADPH oxidase inhibitor in many experimental models. However, concern regarding the efficacy, selectivity, and oxidative side effects of the inhibitor is increasing. In this study, our aim was to characterize the pro-oxidant properties of apocynin and the structurally-related compounds vanillin and vanillic acid. Glutathione (GSH), cysteine, ovalbumin, and the coenzyme NADPH were chosen as potential target biomolecules that could be affected by transient free radicals from apocynin, vanillin and vanillic acid. Additionally, trolox and rifampicin were used as models of hydroquinone moieties, which are particularly susceptible to oxidation. Transient radicals were generated by horseradish peroxidase/hydrogen peroxide-mediated oxidation. In the presence of apocynin, oxidation of GSH was increased seven-fold, and the product of this reaction was identified as GSSG. Similar results were obtained for oxidation of cysteine and ovalbumin. Oxidation of the coenzyme NADPH increased more than 100-fold in the presence of apocynin. Apocynin also caused rapid oxidation of trolox and rifampicin to their quinone derivatives. In conclusion, the pro-oxidant activity of apocynin is related to its previous oxidation leading to transient free radicals. This characteristic may underlie some of the recent findings regarding beneficial or deleterious effects of the phytochemical.     

Interaction of enkephalin derivatives with reactive oxygen species

The oxidation of opioid peptides by tyrosinase in the presence of an excess of a thiol gives rise to cysteinyldopa derivatives. The major products arising from the reaction between Leu-enkephalin and cysteine are represented by 5-S-cysteinyldopaenkephalin (5-CDenk) and 2-S-cysteinyldopaenkephalin (2-CDenk). The interaction of 5-CDenk and 2-CDenk with reactive oxygen species (ROS) has been studied. These compounds are able to scavenge superoxide anion, hydroxyl and peroxyl radicals as well as to reduce the lipid peroxidation rate induced by ABAP. The scavenging activities in all instances are dose-dependent. In some cases CDenks are more active than compounds recognized as strong radical scavengers, such as Trolox and mannitol. As a result of the action of the Fenton system, the CDenks (as well as the Enks) are oxidized into pigmented derivatives. The possible implications of the interaction of CDenks and Enks with ROS on melanization process in Parkinson's disease are discussed.     

Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals.

Thiyl free radicals have been shown to react with polyunsaturated fatty acids via abstraction of bisallylic hydrogen, forming pentadienyl radicals, and via addition to the double bonds. In the absence of oxygen, the latter pathway leads to regeneration of thiyl radicals through beta-elimination or "repair" of the adduct radicals by thiols. In the presence of oxygen, fixation of thiyl-induced damage occurs through reaction of O2 with the pentadienyl radical (yielding conjugated dienyl peroxyl radicals) and also with the thiyl-to-double bond adduct radical. A quantitative reaction scheme evaluated from these data considers abstraction, addition, rearrangement, and repair reactions, and the evaluation of rate constants for the individual steps. Absolute rate constants have been measured, in particular, for reactions of thiyl free radicals from glutathione, cysteine, homocysteine, N-acetylcysteine, cysteine ethyl ester, penicillamine, captopril, mercaptoethanol, and dithiothreitol with polyunsaturated fatty acids (PUFAs) ranging from 18:2 to 22:6, and the lipids trilinolein and trilinolenin. The rate constants for hydrogen abstraction were found to be typically of the order of 10(7) mol-1 dm3 s-1 and to increase with increasing lipophilicity of the attacking thiyl radical. Thioperoxyl radicals, RSOO., were found to be rather unreactive toward PUFAs, in contrast to the isomer sulfonyl radicals, RSO2., which not only abstract hydrogen from the bisallylic methylene groups of the PUFAs (although only at relatively small yield) but also readily add to the PUFA double bonds (major pathway). Specific information was obtained on the optical properties of the thiyl radical derived from the ACE inhibitor captopril, CpS. (lambda max = 340 nm, epsilon = 460 +/- 50 mol-1 dm3 cm-1), and its conjugate disulfide radical anion (CpS:.SCp) (lambda max = 420 nm).     

Conversion of Nitroxide Radicals by Phenolic and Thiol Antioxidants

Nitrone/nitroso spin traps are often used for detection of unstable hydroxyl radical giving stable nitroxide radicals with characteristic electron spin resonance (ESR) signals. This technique may be useful only when the nitroxide radicals are kept stable in the reaction system. The aim of the present study is to clarify whether the nitroxide radicals are kept stable in the presence of the hydroxyl radical scavengers. Effect of hydroxyl radical scavengers on the ESR signals of nitroxide radicals, 2,2,6,6-tetramethyI-piperi-dine-N-oxyl (TEMPO) and the spin adduct (DMPO-OH) of 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and hydroxyl radical, was examined. Although the ESR signals of TEMPO and the DMPO-OH spin adduct were unchanged on treatment with ethanol and dimethyl sulfoxide, their intensities were effectively decreased on treatment with 6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid (Trolox), cysteine, glutathione, 2-mercaptoethanol and metallothionein. Hence, the results of the detection of hydroxyl radical in the presence of phenolic and thiol antioxidants by the ESR technique using nitrone/nitroso spin traps may be unreliable.     

Sonolysis of concentrated aqueous solutions of nonvolatile solutes: spin-trapping evidence for free radicals formed by pyrolysis

The sonolysis of argon-saturated aqueous solutions of sodium acetate, sodium propionate, amino acids, and sugars was investigated by ESR and spin trapping over a large range of concentrations. The spin trap 3,5-dibromo-2,6-dideutero-4-nitrosobenzene sulfonate was used to examine the possibility of detecting new radicals specifically generated in the high temperature zones surrounding the collapsing cavitation bubbles. At lower concentrations of these solutes, no evidence for specific new radicals formed in the high temperature regions induced by cavitation could be found, and only radicals formed by hydroxyl radical and hydrogen atom abstraction reactions could be detected. These were identified by comparison with the radicals produced by uv photolysis in the presence of hydrogen peroxide. However, at higher concentrations, new radicals (typically methyl radicals) formed in the high temperature interfacial regions induced by cavitation were found for sodium acetate, sodium propionate, amino acids, and sugars (D-mannose, D-glucose, 2-deoxy-D-ribose). These results indicate that pyrolysis radicals can be detected when the nonvolatile solutes are present at high concentrations in the interfacial regions of the cavitation bubbles.     

The production of free radicals during the autoxidation of cysteine and their effect on isolated rat hepatocytes

Autoxidizing cysteine has been shown to produce thiyl and hydroxyl radicals. Hydrogen peroxide increased the yield of both radicals which was inhibited by catalase but stimulated by copper / zinc superoxide dismutase. This effect is due to increased bydrogen peroxide production by copper / zinc superoxide dismutase as a result of superoxide dismutation. The production of superoxide radicals could not be detected probably because of its low reactivity, however, measurement of oxygen uptake and reduction of ferricytochrome c by autoxidizing cysteine clearly implicate the involvement of super oxide radicals. The production of hydroxyl radicals is postulated to proceed through a Fenton reaction, however, this may not necessarily be metal ion controlled. Autoxidizing cysteine disrupts the integrity of heptocytes causing release of glutathione, adenosine triphosphate and lactate dehydrogenase indicating that it is of little use as a therapeutic agent.     

Semi-quantitative evaluation of genotoxic activity of chemical substances and evidence for a biological threshold of genotoxic activity

Oxidative DNA damage caused by a cysteine metal-catalyzed oxidation system (Cys-MCO) comprised of Fe(3+), O(2), and a cysteine as an electron donor was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner, as reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks. Unlike CuZnSOD, manganese SOD (MnSOD) as well as iron SOD (FeSOD) did not enhance DNA damage. The capacity of CuZnSOD to enhance damage to DNA was inhibited by a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and a metal chelator, diethylenetriaminepentaacetic acid (DETAPAC). The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with Cys-MCO. We found that the Cys-MCO system caused the release of free copper from CuZnSOD. CuZnSOD also caused the two-fold enhancement of a mutation in the pUC18 lacZ' gene in the presence of Cys-MCO when measured as a loss of alpha-complementation. Based on these results, we interpret the effects of CuZnSOD on Cys-MCO-induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed by the reaction of free Cu(2+), released from oxidatively damaged CuZnSOD, and H(2)O(2) produced by the Cys-MCO system.     

Enhancement of Nitrogen Dioxide-Induced Lipid Peroxidation and Dna Strand Breaking by Cysteine and Glutathione

Nitrogen dioxide less than 100 ppm in air induced lipid peroxidation of liposome composed of l-palmitoyl-2-arachidonylphosphatidylcholine as assessed by thiobarbituric acid reactivity. The nitrogen dioxide-induced lipid peroxidation was enhanced by cysteine, glutathione and bovine serum albumin. While the activity of nitrogen dioxide in air to induce single strand breaks of supercoiled plasmid DNA was low, the breaking was remarkably enhanced by cysteine, glutathione and bovine serum albumin. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that certain strong oxidant(s) were generated by interaction of nitrogen dioxide and cysteine. The spin trapping using 3,5-dibromo-4-nitrosobenzene-sulfonate suggested that sulfur-containing radicals were generated by interaction of nitrogen dioxide and cysteine or glutathione. Hence, certain sulfur-containing radicals generated by the interaction which could effectively induce lipid peroxidation and DNA strand breaks.     

Differences between cysteine and homocysteine in the induction of deoxyribose degradation and DNA damage

The effect of two naturally occurring thiols, such as cysteine and homocysteine, has been examined for their ability to induce deoxyribose degradation and DNA damage. Copper(II) ions have been added to incubation mixtures and oxygen consumption measurements have been performed in order to correlate the observed damaging effects with the rate of metal catalyzed thiol oxidation. Ascorbic acid plus copper has been used as a positive control of deoxyribose and DNA oxidation due to reactive oxygen species. Cysteine or homocysteine in the presence of copper ions induce the degradation of deoxyribose and the yield of 8-hydroxy-2'-deoxyguanosine (8-OHdG), although important differences are observed between the two thiols tested, homocysteine being less reactive than cysteine. DNA cleavage is induced by cysteine in the presence of copper(II) ions but not by homocysteine. Catalase and thiourea, but not superoxide dismutase (SOD), were shown to inhibit the damaging effects of cysteine on deoxyribose or DNA suggesting that H(2)O(2) and *OH radicals are responsible for the observed induced damage. The results indicate that there are differences between the damaging effects of the two thiols tested towards deoxyribose and DNA damage. The pathophysiological importance will be discussed.     

Electrons initiate efficient formation of hydroperoxides from cysteine

Amino acid and protein hydroperoxides can constitute a significant hazard if formed in vivo. It has been suggested that cysteine can form hydroperoxides after intramolecular hydrogen transfer to the commonly produced cysteine sulfur-centered radical. The resultant cysteine-derived carbon-centered radicals can react with oxygen at almost diffusion-controlled rate, forming peroxyl radicals which can oxidize other molecules and be reduced to hydroperoxides in the process. No cysteine hydroperoxides have been found so far. In this study, dilute air-saturated cysteine solutions were exposed to radicals generated by ionizing radiation and the hydroperoxides measured by an iodide assay. Of the three primary radicals present, the hydroxyl, hydrogen atoms and hydrated electrons, the first two were ineffective. However, electrons did initiate the generation of hydroperoxides by removing the –SH group and forming cysteine-derived carbon radicals. Under optimal conditions, 100% of the electrons reacting with cysteine produced the hydroperoxides with a 1:1 stoichiometry. Maximum hydroperoxide yields were at pH 5.5, with fairly rapid decline under more acid or alkaline conditions. The hydroperoxides were stable between pH 3 and 7.5, and decomposed in alkaline solutions. The results suggest that formation of cysteine hydroperoxides initiated by electrons is an unlikely event under physiological conditions.     

Low frequency ultrasound induces aggregation of porcine fumarase by free radicals production

Hydrogen peroxide and hydroxyl-free radicals determine a diffuse aggregation of porcine fumarase and a loss of its enzymatic activity. In this study, hydroxyl-free radicals were generated "in situ" by irradiation with ultrasound (US) at 38 kHz. The structural characteristics of aggregated fumarase were studied using circular dichroism spectroscopy (CD) and steady state fluorescence spectroscopy. Enzyme aggregation is caused by the formation of intermolecular disufide bridges, originated by the oxidation of cysteine residues, together with a diffuse increase in beta-turn in the protein's secondary structure. These conformational changes lead to a fibrous, amyloid-like aggregation which appears ordered and regular under TEM microscopy.     

A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(2)-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO(*) and DMPO in an H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). TEMPO(*) bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H(2)O(2), both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H(2)O(2)-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.