Chemiluminescence study on the peroxidation of linoleic acid initiated by the reaction of ferrous iron with hydrogen peroxide

Linoleic acid was used as a model system to study lipid peroxidation initiated by the reaction of ferrous iron with hydrogen peroxide. Low-level chemiluminescence of the peroxidation was measured with a high-sensitivity single-photon counter. It was found that the luminescence primarily comes from the dimol reaction of singlet oxygen and that the peak intensity of emission is a quadratic function of the concentration of either Fe2+ or H2O2, provided that the other Fenton reagent is in great excess. Under the same conditions, analysis on reaction kinetics shows a linear relationship between the maximal level of the initiator formed by the Fenton reaction and the initial concentration of Fe2+ or H2O2. This implies that the peak intensity of the chemiluminescence may be a good index of the maximal level of the initiator.     

Biological hydroperoxides and singlet molecular oxygen generation

The decomposition of lipid hydroperoxides (LOOH) into peroxyl radicals is a potential source of singlet molecular oxygen ((1)O(2)) in biological systems. Recently, we have clearly demonstrated the generation of (1)O(2) in the reaction of lipid hydroperoxides with biologically important oxidants such as metal ions, peroxynitrite and hypochlorous acid. The approach used to unequivocally demonstrate the generation of (1)O(2) in these reactions was the use of an isotopic labeled hydroperoxide, the (18)O-labeled linoleic acid hydroperoxide, the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) and the direct spectroscopic detection and characterization of (1)O(2) light emission. Using this approach we have observed the formation of (18)O-labeled (1)O(2) by chemical trapping of (1)O(2) with anthracene derivatives and detection of the corresponding labeled endoperoxide by HPLC-MS/MS. The generation of (1)O(2) was also demonstrated by direct spectral characterization of (1)O(2) monomol light emission in the near-infrared region (lambda = 1270 nm). In summary, our studies demonstrated that LOOH can originate (1)O(2). The experimental evidences indicate that (1)O(2) is generated at a yield close to 10% by the Russell mechanism, where a linear tetraoxide intermediate is formed in the combination of two peroxyl radicals. In addition to LOOH, other biological hydroperoxides, including hydroperoxides formed in proteins and nucleic acids, may also participate in reactions leading to the generation (1)O(2). This hypothesis is currently being investigated in our laboratory.     

Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin.

Using 4-methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter- and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent. With 4-trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron-transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate-limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C-H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron-transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.     

Transient formation of the oxo–iron(IV) porphyrin radical cation during the reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin with hydrogen peroxide in aqueous solution

The reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin (Fe(III)TMPyP) with hydrogen peroxide (H₂O₂) and the catalytic activity of the reaction intermediates on the luminescent peroxidation of luminol in aqueous solution were studied by using a double‐mixing stopped‐flow system. The observed luminescence intensities showed biphasic decay depending on the conditions. The initial flashlight decayed within <1 s followed by a sustained emission for more than 30 s. Computer deconvolution of the time‐resolved absorption spectra under the same conditions revealed that the initial flashlight appeared during the formation of the oxo–iron(IV) porphyrin, TMPyPFe(IV) = O, which is responsible for the sustained emission. The absorption spectra 0.0–0.5 s did not reproduce well by a simple combination of the two spectra of Fe(III)TMPyP and TMPyPFe(IV) = O, indicating that transient species was formed at the initial stage. Addition of uric acid (UA) caused a significant delay in the initiation of the luminol emission as well as in the formation of the TMPyPFe(IV) = O. Both of them were completely diminished in the presence of UA equimolar with H₂O₂, while mannitol had no effect at all. The delay of the light emission as well as the appearance of TMPyPFe(IV) = O was directly proportional to the [UA]₀ but other kinetic profiles were not changed significantly. Based on these observations and the kinetic analysis, we confirmed the involvement of the oxo–iron(IV) porphyrin radical cation, (TMPyP)^·+Fe(IV) = O, as an obligatory intermediate in the rate‐determining step of the overall reaction, Fe(III)TMPyP + H₂O₂ → TMPyPFe(IV) = O, with a rate constant of k = 4.3 × 10⁴/mol/L/s. The rate constants for the reaction between the (TMPyP)^·+Fe(IV) = O and luminol, and between the TMPyPFe(IV) = O and luminol were estimated to be 3.6 × 10⁶/mol/L/s and 1.31 × 10⁴/mol/L/s, respectively. Copyright © 2003 John Wiley & Sons, Ltd.     

Experimental study on photodynamic diagnosis of cancer mediated by chemiluminescence probe

A novel method of photodynamic diagnosis of cancer mediated by chemiluminescence probe is presented. The mechanism for photodynamic therapy involves singlet oxygen ((1)O(2)) generated by energy transfer from photosensitizers. (1)O(2) can react with 3,7-dihydro-6-[4-[2-(N'-(5-fluoresceinyl)thioureido)ethoxy]phenyl]-2-methylimidazo[1,2-a]pyrazin-3-one sodium salt (FCLA), which is a Cypridina luciferin analog and a specific chemiluminescence probe for detecting (1)O(2) and superoxide (O(2)(-)). The reaction of FCLA and (1)O(2) can give emission with peak wavelength at about 532 nm. In the present study, FCLA was chosen as an optical reporter of (1)O(2) produced from the photosensitization reaction of hematoporphyrin derivative in model solution and in nude mice with transplanted mammary cancer. Photosensitized chemiluminescence from the reaction of FCLA with (1)O(2) was detected by a highly sensitive Intensified Charge-Coupled Device detector. The chemiluminescence was markedly inhibited by the addition of 10 mmol/l sodium azide (NaN(3)) to the model solution and minor effects were observed at the addition of 10 micromol/l superoxide dismutase, 20 mmol/l mannitol and 100 microg/ml catalase, respectively, thus indicating that (1)O(2) generation from photosensitization reaction mainly results in light emission. Experiments in vivo with tumor-bearing mice showed a clear chemiluminescence image of tumor. The study suggests that this novel method may be applicable to the diagnosis of superficial tumors.     

Chemically shifted singlet oxygen spectrum.

The estimated light emission spectrum was determined for a singlet oxygen (1O2)-producing system, NaOCl + H2O2, alone and in the presence of tryptophan and bovine serum albumin. Tryptophan and bovine serum albumin caused a decrease in the red emission of 1O2 and an increase in the amount of shorter wavelength light. This effect was due to chemiluminescence rather than fluorescence. Arachidonic acid caused a similar spectral shift, while guanosine demonstrated a late chemiluminescent reaction of predominantly short wavelength light in the presence of 1O2.     

Chemiluminescence of the peroxomonosulphate-cobalt(II)-aliphatic monocarboxylic acids system.

A weak chemiluminescence (CL) emission was observed due to the production of singlet oxygen ((1)O(2)) during the decomposition of peroxomonosulphate (HSO(5)(-)) catalysed by cobalt(II). Low molecular mass aliphatic monocarboxylic acids, such as formic, acetic, propionic, butyric and valeric acids, influenced the CL emission, and the reaction of aliphatic monocarboxylic acids with HSO(5)(-)/Co(2+) solution was further investigated using a flow injection analysis (FIA) CL method. The results indicated that the CL intensities of aliphatic monocarboxylic acids were improved with increase in the carbon chain length in the potassium peroxomonosulphate-cobalt(II) sulphate system. Generation of singlet oxygen was confirmed by the fact that the CL emission of aliphatic monocarboxylic acids with the HSO(5)(-)/Co(2+) solution was quenched by NaN(3), and from the CL spectrum of the reaction system. Additionally, a possible mechanism of aliphatic monocarboxylic acids CL emission enhancement was proposed.     

Cytochrome P-450-mediated oxidation of substrates by electron-transfer; role of oxygen radicals and of 1- and 2-electron oxidation of paracetamol

The mechanism by which the hepatic cytochrome P-450 (Cyt. P-450) containing mixed-function oxidase system oxidizes the analgesic drug paracetamol (PAR) to a hepatotoxic metabolite was studied. Since previous studies excluded the possibility of oxygenation of PAR, three other mechanisms, namely direct 1-electron oxidation by a Cyt. P-450-ferrous-dioxygen complex under concomitant formation of H2O2 to N-acetyl-p-semiquinone imine (NAPSQI), direct 2-electron oxidation by a Cyt. P-450-ferric-oxene complex to N-acetyl-p-benzoquinone imine (NAPQI) and indirect oxidation by active oxygen species released from Cyt. P-450, were considered. Indirect oxidation by active oxygen species was not involved, as active oxygen scavengers such as superoxide dismutase, catalase and DMSO did not affect the oxidation of PAR in hepatic microsomes. No reaction products characteristic for a direct 1-electron oxidation of PAR by Cyt. P-450 were observed: neither NAPSQI radical formation was detectable by ESR, nor PAR-dimer formation, nor stimulation of the microsomal H2O2 production was found to occur. In fact, PAR inhibited the spontaneous microsomal H2O2 formation. Studies on the reactions of NAPSQI with glutathione (GSH) revealed that NAPSQI hardly conjugated with GSH to a 3-glutathionyl-paracetamol conjugate (PAR-GSH) conjugate. The reactions of the elusive reactive metabolite formed during microsomal oxidation of PAR in the presence of GSH closely resembled those of synthetic NAPQI: both PAR-GSH and oxidized glutathione (GSSG) formation occurred. Furthermore, in agreement with a 2-electron oxidation hypothesis, iodosobenzene-dependent oxidation of PAR by cyt. P-450 in the presence of GSH resulted in the formation of the PAR-GSH conjugate. It is concluded that bioactivation of PAR by the Cyt. P-450 containing mixed-function oxidase system consists of a direct 2-electron oxidation to NAPQI.     

Exposure of nuclear medicine patients to ionizing radiation is associated with rises in HPRT- mutant frequency in peripheral T-lymphocytes

When Chinese hamster fibroblasts were exposed to hydrogen peroxide or to a system consisting of xanthine oxidase and hypoxanthine, which generates superoxide anion plus hydrogen peroxide, sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner. When the iron-complexing agent o-phenanthroline was present in the medium, however, the production of these SCEs was completely inhibited. This fact indicates that the Fenton reaction: Fe2+ + H2O2----OH0 + OH- + Fe3+ is responsible for the production of SCEs. When O2- and H2O2 were generated inside the cell by incubation with menadione, the production of SCE was prevented by co-incubation with copper diisopropylsalicylate, a superoxide dismutase mimetic agent. The most likely role of O2- is as a reducing agent of Fe3+: O2- + Fe3+----Fe2+ + O2, so that the sum of this and the Fenton reaction, i.e., the iron-catalyzed Haber-Weiss reaction, provides an explanation for the active oxygen species-induced SCE: H2O2 + O2(-)----OH- + OH0 + O2. According to this view, the OH radical thus produced is the agent which ultimately causes SCE. These results are discussed in comparison with other mechanisms previously proposed for induction of SCE by active oxygen species.     

Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells

Singlet oxygen (1O2), a reactive oxygen species, has been found to be implicated in many cellular events and pathological disorders. Herein, we investigated the reactivity of 1O2 towards the anaesthetic agent propofol (PPF) encapsulated within DMPC liposomes. By time resolved luminescence, the rate constant of 1O2 quenching by PPF was evaluated, depending on the location of the sensitizer, with following values: 1.35+/-0.05x10(7) M(-1) s(-1) for deuteroporphyrin (as embedded source) and 0.8+/-0.04x10(7) M(-1) s(-1) for uroporphyrin (as external source), respectively. The nature of the oxidation product, resulting from the reaction of 1O2 with PPF, was determined using absorption and HPLC techniques. Finally, the in vitro protective effect of PPF towards the 1O2-induced neuronal cell toxicity was evaluated in terms of cell viability.     

Formation of a diimino-imidazole nucleoside from 2'-deoxyguanosine by singlet oxygen generated by methylene blue photooxidation

Singlet oxygen ((1)O(2)) is capable of inducing genotoxic, carcinogenic and mutagenic effects. It has previously been reported that the reaction of (1)O(2) with 2'-deoxyguanosine, which is a major target of (1)O(2) among the DNA constituents, leads to formation of various oxidized products including 8-oxo-7,8-dihydro-2'-deoxyguanosine and spiroiminodihydantoin, amino-imidazolone and diamino-oxazolone nucleosides. In addition to these products, we report that a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dD), is formed by reaction of 2'-deoxyguanosine with (1)O(2) generated by irradiation with visible light in the presence of methylene blue under aerobic conditions. Its identification is based on identical chromatographic and spectroscopic data with an authentic compound, which we recently isolated and characterised from the reaction mixture of 2'-deoxyguanosine with reagent HOCl and a myeloperoxidase-H(2)O(2)-Cl(-) system. The yield of dD was increased by D(2)O and decreased by azide. dD was not generated from 8-oxo-7,8-dihydro-2'-deoxyguanosine. These results indicate that dD is generated by (1)O(2) directly from 2'-deoxyguanosine, but not via 8-oxo-7,8-dihydro-2'-deoxyguanosine. dD may play a role in the genotoxicity of singlet oxygen in cells.     

Loss of transforming activity of plasmid DNA (pBR322) in E. coli caused by singlet molecular oxygen

Plasmid DNA pBR322 in aqueous solution was exposed to singlet molecular oxygen (1O2) generated by microwave discharge. DNA damage was detected as loss of transforming activity of pBR322 in E. coli (CMK) dependent on the time of exposure. DNA damage was effectively decreased by singlet-oxygen quenchers such as sodium azide and methionine. Replacement of water in the incubation buffer by D2O led to an increase in DNA damage. 9,10-Bis(2-ethylene)anthracene disulfate was used as a chemical trap for 1O2 quantitation by HPLC analysis of the endoperoxide formed.     

Highly-sensitive electrochemical immunosensing method based on dual amplification systems

In order to rapidly and simultaneously quantify and screen trace levels of multiple biomarkers in a single sample, rapid 1,1'-oxalyldiimidazole chemiluminescence (ODI CL) was applied as a biosensor of immunoassays using various enzymes such as alkaline phosphatase (ALP) and horseradish peroxidise (HRP). (1) Fluorescein was formed from the reaction of fluorescein diphosphate (FDP) and immuno-complex conjugated with ALP. (2) Resorufin was formed from the reaction between Amplex Red and H(2)O(2) in the presence of immuno-complex conjugated with HRP. When ODI CL reagents (H(2)O(2) in isopropyl alcohol, ODI in ethyl acetate) were injected in a test tube or strip-well containing fluorescein and resorufin formed from above two reactions a bright CL emission spectrum having two peaks (518 nm for fluorescein and 602 nm for resorufin) was observed. The two peaks can be independently quantified with an appropriate statistical tool capable of deconvoluting multiple emission peaks. In conclusion, we expect that ODI chemiluminescent enzyme immunoassays (CLEIAs) using a couple of enzymes conjugated with antigen or antibody and substrates can rapidly and simultaneously quantify and screen multiple biomarkers in a single sample.     

Stopped-flow kinetic analysis of the bacterial luciferase reaction.

The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. Luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The time courses for the formation and decay of the various intermediates have been followed by monitoring the absorbance changes at 380 and 445 nm along with the emission of visible light using n-decanal as the alkyl aldehyde. The synthesis of the 4a-hydroperoxyflavin intermediate (FMNOOH) was monitored at 380 nm after various concentrations of luciferase, O2, and FMNH2 were mixed. The second-order rate constant for the formation of FMNOOH from the luciferase-FMNH2 complex was found to be 2.4 x 10(6) M-1 s-1. In the absence of n-decanal, this complex decays to FMN and H2O2 with a rate constant of 0.10 s-1. The enzyme-FMNH2 complex was found to isomerize prior to reaction with oxygen. The production of visible light reaches a maximum intensity within 1 s and then decays exponentially over the next 10 s. The formation of FMN from the intermediate pseudobase (FMNOH) was monitored at 445 nm. This step of the reaction mechanism was inhibited by high levels of n-decanal which indicated that a dead-end luciferase-FMNOH-decanal could form. The time courses for these optical changes have been incorporated into a comprehensive kinetic model. Estimates for 15 individual rate constants have been obtained for this model by numeric simulations of the various time courses.     

Formation of an oxo-radical of peroxovanadate during reduction of diperoxovanadate with vanadyl sulfate or ferrous sulfate

Formation of oxygen radicals during reduction of H(2)O(2) or diperoxovanadate with vanadyl sulfate or ferrous sulfate was indicated by the 1:2:2:1 electron spin resonance (ESR) signals of the DMPO adduct typical of standard ()OH radical. Signals derived from diperoxovanadate remained unchanged in the presence of ethanol in contrast to those from H(2)O(2). This gave the clue that they represent a different radical, possibly (*)OV(O(2))(2+), formed on breaking a peroxo-bridge of diperoxovanadate complex. The above reaction mixtures evolved dioxygen or, when NADH was present, oxidized it rapidly which was accompanied by consumption of dioxygen. Operation of a cycle of peroxovanadates including this new radical is suggested to explain these redox activities both with vanadyl and ferrous sulfates. It can be triggered by ferrous ions released from cellular stores in the presence of catalytic amounts of peroxovanadates.     

Mechanistic implications of variable stoichiometries of oxygen consumption during tyrosinase catalyzed oxidation of monophenols and o-diphenols

The stoichiometry of oxygen consumption during tyrosinase-catalyzed oxidation of an o-diphenol (4-tert-butylcatechol, TBC) and a monophenol (4-tert-butylphenol, TBP) has been determined. At high [substrate]/[enzyme] ratios, in the case of o-diphenols, the stoichiometry of the enzyme-catalyzed reaction was always 1 O(2)/2 o-diphenols, although if the o-quinone product was unstable, the apparent stoichiometry could tend to 1 O(2)/1 o-diphenol due to regeneration of an o-diphenol in a side reaction. In the case of monophenols, the stoichiometry could be 1 O(2)/1 monophenol or 1.5 O(2)/1 monophenol depending if the o-quinone product was stable or unstable, respectively. However, at low [substrate]/[enzyme] ratios, the oxygen/substrate stoichiometry could, even in the case where stable products are formed, be lower than 1 O(2)/2 substrates for o-diphenols or higher than 1 O(2)/1 substrate for monophenols. These data supported the mechanism proposed by Rodríguez-López et al. [J. Biol. Chem. 267 (1992) 3801-3810], in which, during hydroxylation of monophenols, tyrosinase first transformed monophenol to o-diphenol and then either catalyzed a further oxidation to form o-quinone or released it into the reaction medium. In this second case, subsequent oxidation of the o-diphenol resulted in additional oxygen consumption.     

Myeloperoxidase singlet molecular oxygen generation detected by direct infrared electronic emission

Myeloperoxidase in micromolar concentrations reacting with half-millimolar stock solution H2O2 in acetate buffer containing KBr and in 50% D2O (pH + pD = 4.5) at 298 K is shown to generate singlet delta molecular oxygen efficiently. The near infrared electronic emission of singlet oxygen at 1268 nm is detected directly by novel ultrasensitive IR spectrophotometer equipment. The quantum efficiency of singlet oxygen generation by the MPO X Br- X H2O2 reaction is shown to be comparable with that of the standard chemical reaction OCl- X H2O2 at identical peroxide concentrations.     

Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.     

The Haber-Weiss cycle--70 years later

The chain reactions HO* + H2O2 --> H2O + O2*- + H+ and O2*- + H+ + H2O2 --> O2 + HO* + H2O, commonly known as the Haber-Weiss cycle, were first mentioned by Haber and Willst?tter in 1931. George showed in 1947 that the second reaction is insignificant in comparison to the fast dismutation of superoxide, and this finding appears to have been accepted by Weiss in 1949. In 1970, the Haber-Weiss reaction was revived by Beauchamp and Fridovich to explain the toxicity of superoxide. During the 1970s various groups determined that the rate constant for this reaction is of the order of 1 M(-1) s(-1) or less, which confirmed George's conclusion. The reaction of superoxide with hydrogen peroxide was dropped from the scheme of oxygen toxicity, and superoxide became the source of hydrogen peroxide, which yields hydroxyl radicals via the Fenton reaction, Fe2+ + H2O2 --> Fe3+ + HO- + HO*. In 1994, Kahn and Kasha resurrected the Haber-Weiss reaction again, but this time the oxygen was believed to be in the singlet (1delta(g)) state. As toxicity arises not from a Fenton-catalysed Haber-Weiss reaction, but from the Fenton reaction, the Haber-Weiss reaction should not be mentioned anymore.     

Reaction of disodium cromoglycate with hydrated electrons

A possible mechanism by which disodium cromoglycate (DSCG) prevents a decrease in regional cerebral blood flow but not hypotension in primates following whole body gamma-irradiation was studied. Several studies have implicated superoxide radicals (O2-.) in intestinal and cerebral vascular disorders following ischemia and ionizing radiation, respectively. O2-. is formed during radiolysis in the reaction between hydrated electrons (e-aq) and dissolved oxygen. For this reason, the efficiency of DSCG to scavenge e-q and possibly prevent the formation of O2-. was studied. Hydrated electrons were produced by photolysis of potassium ferrocyanide solutions. The rate constant, k = 2.92 x 10(10) M-1s-1 for the reaction between e-aq and DSCG was determined in competition experiments using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). This spin trap reacts rapidly with e-aq followed by protonation to yield the ESR observable DMPO-H spin adduct. The results show that DSCG is an efficient e-aq scavenger and may effectively compete with oxygen for e-aq preventing the radiolytic formation of O2-..