On the reaction of molecular oxygen with thiyl radicals: a re-examination

Absolute rate constants for the addition of oxygen to thiyl radicals, i.e. RS. + O2----RSOO., have been determined by applying a new competition method based on RS. formation via one-electron reduction of the corresponding disulphides, and the competition between RS. reacting with O2 and an electron donor such as ascorbate. Bimolecular rate constants have been obtained for the thiyl radicals derived from cysteine (6.1 X 10(7) mol-1 dm3 s-1), penicillamine (2.5 X 10(7) mol-1 dm3 s-1), homocysteine (8.0 X 10(7) mol-1 dm3 s-1), cysteamine (2.8 X 10(7) mol-1 dm3 s-1), 3-thiopropionic acid (2.2 X 10(8) mol-1 dm3 s-1) and glutathione (3.0 X 10(7) mol-1 dm3 s-1), respectively. The values obtained for the O2 addition to the thiyl radicals from glutathione and cysteine are considerable lower (by about two orders of magnitude) than those previously published. This indicates that the RS. + O2 reaction may be of complex nature and is generally a process which is not solely controlled by the diffusion of the reactants.     

Interactions of thiyl free radicals with oxygen: a pulse radiolysis study

A pulse radiolysis study of glutathione in aqueous solution at pH 5.5 containing N2O/O2 mixtures at various ratios indicates that oxygen rapidly adds to the thiyl glutathione radical yielding a transient absorption, with a maximum at 540 nm, whose characteristics appear to be compatible with assignment to the GSOO. radical. The reaction (Formula: see text) appears to be an equilibrium whose kinetic constants have been estimated (kf = 2.0 X 10(9) dm3 mol-1, kb = 6.2 X 10(5) s-1). Evidence for electron transfer from ascorbate to the GSOO. radical has been obtained and the respective rate constant has been determined to be 1.75 +/- 0.15 X 10(8) dm3 mol-1 s-1.     

Electron spin resonance and pulse radiolysis studies on the spin trapping of sulphur-centered radicals

Thiyl radicals are shown to be readily trapped with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO) giving characteristic spin adducts with hyperfine coupling constants aN 1.52-1.58, aH 1.52-1.80 mT, and g values in the range 2.0065-2.0067 for the DMPO adducts and aN 1.50-1.56, aH 1.70-1.92 mT, g 20049-2.0051 for the TMPO adducts. Kinetic data obtained from pulse radiolysis studies show that, in general, thiyl radicals react rapidly with these spin traps with rate constants of the order of 10(7)-10(8) dm3 mol-1 s-1. The tetramethylated spin trap TMPO though giving slightly less intense electron spin resonance (ESR) spectra, produces longer lived adducts, and is suggested to be of greater utility due to the more characteristic nature of the coupling constants of the observed adducts; reaction of certain thiyl radicals with DMPO produces adducts which are superficially similar to the hydroxyl radical adduct to the same trap.     

The reverse of the 'repair' reaction of thiols: H-abstraction at carbon by thiyl radicals

Thiyl radicals (RS) formed by the reaction of radiolytically generated OH radicals with thiols, e.g. 1,4-dithiothreitol (DTT), react with cis- and trans-2,5-dimethyltetrahydrofuran by abstracting an H atom in the alpha-position to the ether function (k approximately equal to 5 X 10(3) dm3 mol-1 s-1). The so-formed planar ether radical is 'repaired' by the thiol (k = 6 X 10(8) dm3 mol-1 s-1) thereby regenerating a cis- or trans-2,5-dimethyltetrahydrofuran molecule. In this reaction a thiyl radical is reproduced. Thus trans-2,5-Me2THF from cis-2,5-Me2THF and vice versa are formed in a chain reaction: at a dose rate of 2.8 X 10(-3) Gys-1 and a trans-2,5-Me2THF concentration of 1 X 10(-2) mol dm-3 using DTT as the thiol, G(cis-2,5-Me2THF) = 160 has been found. The chain reaction is very sensitive to impurities and also to disulphides such as those radiolytically formed. 2,5-Me2THF can be regarded as a model for the sugar moiety of DNA where the C(4')-radical is known to lead to DNA strand breakage. The possible role of cellular thiols in the repair of the C(4') DNA radical, and also the conceivable role of thiyl radicals inducing DNA strand breakage, are discussed.     

Thiyl radicals abstract hydrogen atoms from carbohydrates: reactivity and selectivity.

Free radical damage of DNA is a well-known process affecting biological tissue under conditions of oxidative stress. Though carbohydrate-derived radicals are generally "repaired" by hydrogen transfer from thiols, the reverse possibility, namely hydrogen abstraction by thiyl radicals from carbohydrates, exists. The biological relevance of this process has been discussed controversially, especially because of the lack of rate constants. Therefore, we have measured rate constants for the hydrogen transfer reaction between thiyl radicals from cysteine and selected carbohydrates, 2-deoxy-D-ribose (dRib), 2-deoxy-D-glucose (dGls), alpha-D-glucose (Gls), and inositol (Ino). Rate constants are on the order of 10(4) M(-1)s(-1), with the highest average value for dRib, (2.7 +/- 1.0) x 10(4) M(-1)s(-1), and the lowest average value for dGls, (1.6 +/- 0.2) x 10(4) M(-1)s(-1), based on two ways of kinetic analysis, standard competition kinetics and stochastic simulation of the experimental results, respectively. In general, thiyl radicals attack preferentially the C(1)-H bond of the carbohydrates, to an extent of ca. 72% in dRib and 90% in dGls. Kinetic measurements were possible through a specifically designed competition system measuring the reaction of thiyl radicals with either the C-H bonds of the carbohydrates or the C(alpha)-H bond of cysteine under conditions where the extent of other competitive reactions of the thiyl radicals were minimized.     

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.     

Sulfinyl radical formation from the reaction of cysteine and glutathione thiyl radicals with molecular oxygen

Using Electron Spin Resonance spectroscopy at low temperatures, we find that thiyl radicals resulting from irradiation of frozen aqueous solutions of a variety of thiols, including cysteine, glutathione, and penicillamine react with oxygen to form sulfinyl (RSO.) radicals. The identity of the cysteine sulfinyl radical has been confirmed by the use of molecular oxygen isotopically labeled with 17O. Previous workers have suggested the reaction of thiyl radicals and molecular oxygen resulted in the formation of the potentially damaging thiol peroxyl radical, RSOO.; our work shows no evidence for this species. The sulfinyl radicals are suggested to result from a direct reaction between thiyl radicals and molecular oxygen. This reaction results in the cleavage of the dioxygen bond.     

Spin trapping the cysteine thiyl radical with phenyl-N-t-butylnitrone

The cysteine thiyl radical has been detected in a variety of biological systems by means of the ESR spectrum of the adduct between the radical and nitrone spin traps. 5,5-Dimethyl-1-pyroline N-oxide (DMPO) is the spin trap of choice in these studies for several reasons. However, we show here that the adduct between the cysteine thiyl radical and phenyl-N-t-butylnitrone (PBN) spin trap can be observed under certain oxidizing conditions where the adduct with DMPO is not detected. This suggests the use of PBN in searching for the thiyl radical under such conditions.     

Repair of amino acid radicals by a vitamin E analogue

Free radicals derived from one-electron oxidation of the amino acids tryptophan, tyrosine, methionine and histidine have been found to be rapidly (k = 10(7) -10(9) dm3 mol-1 s-1) and efficiently repaired by Trolox C, a vitamin E analogue. The reactions form a relatively stable phenoxyl radical of Trolox C (lambda max = 440 nm; epsilon = 5.4 X 10(3) mol dm-3 cm-1). The radical cation of tryptophan is more rapidly repaired than the neutral tryptophan radical. Repair of tryptophanyl radicals in the enzyme lysozyme has also been observed. The results suggest that a function of alpha-tocopherol in membranes may be the repair of radicals of integral membrane proteins.     

Proteins protect lipid membranes from oxidation by thiyl radicals

Oxidation of polyunsaturated fatty acids by thiyl radicals derived from GSH or Cys is believed to be responsible for some of the biological damage resulting from lipid oxidation under oxidative stress. However, this has not been demonstrated in complex biological systems. In this study, we measured the formation of lipid hydroperoxides in liposomes exposed to radicals generated by gamma radiation from GSH, GSSG, GSMe, Cys and Met. In the absence of proteins, the radicals oxidized the liposome lipids. In the presence of proteins, the thiyl radicals failed to react with the liposomes, even though the protein radicals efficiently oxidized the S-compounds. It appears that the thiyl and other S-radicals were effectively scavenged by the protein before initiating lipid oxidation. The results suggest that membrane lipid oxidation in vivo by thiyl radicals is unlikely to be a significant event.     

Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.

In the Escherichia coli class Ia ribonucleotide reductase (RNR), the best characterized RNR, there is no spectroscopic evidence for the existence of the postulated catalytically essential thiyl radical (R-S(*)) in the substrate binding subunit R1. We report first results on artificially generated thiyl radicals in R1 using two different methods: chemical oxidation by Ce(IV)/nitrilotriacetate (NTA) and laser photolysis of nitric oxide from nitrosylated cysteines. In both cases, EPR spin trapping at room temperature using phenyl-N-t-butylnitrone, and controls with chemically blocked cysteines, has shown that the observed spin adduct originates from thiyl radicals. The EPR line shape of the protein-bound spin adduct is typical for slow motion of the nitroxide moiety, which indicates that the majority of trapped thiyl radicals are localized in a folded region of R1. In aerobic R1 samples without spin trap that were frozen after treatment with Ce(IV)/NTA or laser photolysis, we observed sulfinyl radicals (R-S(*)=O) assigned via their g-tensor components 2.0213, 2.0094, and 2.0018 and the hyperfine tensor components 1.0, 1.1, and 0.9 mT of one beta-proton. Sulfinyl radicals are the reaction products of thiyl radicals and oxygen and give additional evidence for generation of thiyl radicals in R1 by the procedures used.     

Reactions of linoleic acid peroxyl radicals with phenolic antioxidants: a pulse radiolysis study

Linoleic acid peroxyl radicals (LOO.) can be viewed as model intermediates occurring during lipid peroxidation processes. Formation and reactions of these species were investigated in aqueous alkaline solution using the technique of pulse radiolysis combined with kinetic spectroscopy. Irradiation of linoleic acid in N2O/O2-saturated solutions leads to a mixture of peroxyl radical isomers, whereas reaction of 13-hydroperoxylinoleic acid (13-LOOH) with azide radicals in N2O-saturated solution produces 13-LOO. radicals specifically. These peroxyl radicals cannot be observed directly, but their reactions with the two flavonols, kaempferol and quercetin, acting as radical-scavenging antioxidants, produced strongly absorbing aroxyl radicals (ArO.). The same aroxyl radicals were generated by .OH and N3. with rate constants exceeding 10(9) dm3 mol-1 s-1. Applying a reaction scheme that includes competing generation and decay reactions of both LOO. and ArO. radicals, we derived individual rate constants for LOO. reactions with the phenols (greater than 10(7) dm3 mol-1 s-1), with the aroxyl radicals to form covalent adducts (greater than 10(8) dm3 mol-1 s-1), as well as for their bimilecular decay (3.0 X 10(8) dm3 mol-1 s-1). These results demonstrate the high reactivity of both fatty acid peroxyl radicals and the flavone antioxidants in aqueous solution.     

In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.     

Hypochlorite-induced oxidation of thiols: Formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Hypochlorite-induced oxidation of thiols: formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Reactions of captopril and epicaptopril with transition metal ions and hydroxyl radicals: An EPR spectroscopy study

In the present study, using the technique of EPR spin trapping with DMPO a spin trap, we demonstrated formation of thiyl radicals from thiol-containing angiotensin converting enzyme (ACE) inhibitor captopril (CAP) and from its stereoisomer epicaptopril (EPICAP), a non-ACE inhibitor, in the process of ^.OH radical scavenging. Splitting constants of DMPO/thiyl radical adducts were identical for both thiols and were a_N = 15.3 G, and a_H = 16.2 G. Bimolecular rate constants for the reaction of CAP and EPICAP with ^.OH radicals were close to a diffusion-controlled rate (≈ 2 × 10¹⁰ M⁻¹s⁻¹). Our data also show that both CAP and EPICAP reduce Fe(III) ions and that their respective thiyl radicals are formed in this reaction. In the presence of Fe(III), H₂O₂, and CAP, or EPICAP, ^.OH radicals were produced by a thiol-driven Fenton mechanism. Copper(II) ions were also reduced by these thiols, but no thiyl radicals could be detected in these reactions, and no ^.OH or other Fenton oxidants were observed in the presence of H₂O₂. Our data show direct evidence that thiol groups of CAP and EPICAP are involved in scavenging of ^.OH radicals. The direct ^.OH radical scavenging, together with the reductive “repair” of other sites of ^.OH radical attack, may contribute to the known protective effect of CAP against ischemia/reperfusion-induced arrhythmias. The formation of reactive thiyl radicals in the reactions of the studied compounds with ^.OH radicals and with Fe(III) ions may play a role in some of the known adverse effects of CAP.     

Observations on the reactivity of thiyl radicals derived from 3,6-epidithiodiketopiperazine-2,5-diones and related congeners

A range of thiyl radicals derived from the reduced form of epidithiodiketopiperazines (ETPs) act as polarity reversal catalysts for the hydrosilylation of an enol lactone but not for H-atom abstraction from a model ribose ester.     

Thiol peroxyl radical formation from the reaction of cysteine thiyl radical with molecular oxygen: an ESR investigation

Using Electron Spin Resonance (ESR) spectroscopy, we have identified the cysteine thiol peroxyl radical (CysSOO.) at low temperatures in two aqueous glasses. This radical shows a typical peroxyl radical ESR spectrum, but unlike carbon-based peroxyl radicals has a violet color (lambda max = 540 nm) and forms a new radical showing a singlet ESR spectrum when photobleached with visible light. The cysteine peroxyl radical reacts to form the cysteine sulfinyl radical (CysSO.) in the glass which allows warming to 165K. 17O isotopic substitution studies indicate dissolved molecular oxygen is the source of oxygen in CysSOO.. Anisotropic g-values and the parallel anisotropic 17O hyperfine couplings for this radical are reported.     

Pulse radiolytic studies of radicals produced from methylated uracils via oxidation by SO4.

Using a pulse radiolysis technique, some nucleic base radicals were produced by the reactions of sulfate radical, SO4-, with 1-, 3-, 5-, and 6-methyluracils, and their optical and kinetic natures were observed. All of their absorption spectra showed main peaks at approximately 400 nm with absorption constants ranging from 1020 to approximately 1560 dm3 mol-1 cm-1. The rate constants of their formation were 1.6 to approximately 3.3 X 10(9) dm3 mol-1 s-1. For thymine and 6-methyluracil, the absorption coefficients of their radicals at approximately 500 nm changed according to pH, giving pK values of approximately 9. For N(3)-methylated uracil, on the other hand, no such acid-base equilibrium was found. When the N(1) position was methylated, another type of pH effect was found. From these spectral observations and the comparative discussions, it was shown that methylation at the N(1) position gives OH-adduct radicals and at other positions proton-released radicals. For 3- and 6-methyluracils, second intermediates were formed concomitantly with the disappearance of the initial radicals. They are tentatively assigned to their ring-opened radicals, presumably by the reaction of the initial radicals with S2O8(2-).     

Enzyme-bound pentadienyl and peroxyl radicals in purple lipoxygenase

Samples of purple lipoxygenase prepared by addition of either 13-hydroperoxy-9,11-octadecadienoic acid or linoleic acid and oxygen to ferric lipoxygenase contain pentadienyl and/or peroxyl radicals. The radicals are identified by the g values and hyperfine splitting parameters of natural abundance and isotopically enriched samples. The line shapes of their EPR spectra suggest the radicals are conformationally constrained when compared to spectra of the same radicals generated in frozen linoleic acid. Further, the EPR spectra are unusually difficult to saturate. The radicals are stable in buffered aqueous solution at 4 degrees C for several minutes. All of this implies that these species are bound to the enzyme, possibly in proximity to the iron. Only peroxyl radical is seen when the purple enzyme is generated with either hydroperoxide or linoleic acid in O2-saturated solutions. Addition of natural abundance hydroperoxide under 17O-enriched O2 leads to the 17O-enriched peroxyl radical, while the opposite labeling results in the natural abundance peroxyl radical, demonstrating the exchange of oxygen. Both radicals are detected in samples of purple lipoxygenase prepared with either linoleic acid or hydroperoxide under air. Addition of the hydroperoxide in the absence of oxygen favors the pentadienyl radical. We propose that addition of either linoleic acid or hydroperoxide to ferric lipoxygenase leads to multiple mechanistically connected enzyme complexes, including those with (hydro)peroxide, peroxide, peroxyl radical, pentadienyl radical, and linoleic acid bound. This hypothesis is essentially identical with the proposed radical mechanism of oxygenation of polyunsaturated fatty acids by lipoxygenase.