化能自养硫氧化细菌Halothiobacillus sp.LS2介导的以乙炔为电子受体的硫氧化反应

【目的】探究化能自养硫氧化细菌Halothiobacillus sp. LS2介导的以乙炔为电子受体的厌氧硫氧化反应。【方法】稀释涂布法测定细胞生长情况,离子色谱仪测试硫氧化动力学中SO_4~(2–)和S_2O_3~(2–)以及基于相对荧光定量法的基因表达分析。【结果】尽管菌株LS2在以氧气为电子受体时的最大反应速率V_(max)更高,但在厌氧条件下且以乙炔为电子受体时,菌株LS2的生长量是氧气为电子受体时的2倍,且硫氧化酶基因soxB的表达量显著高于氧气作为电子受体时。【结论】菌株LS2不仅可以以乙炔为电子受体完成厌氧硫氧化反应,且这一代谢过程的产能效率较有氧硫氧化过程更高。本研究首次发现了微生物介导的以乙炔为电子受体的厌氧硫氧化反应,对丰富硫的生物地球化学循环理论有积极意义。     

Nitroxide-Stimulated H2O2 Decomposition by Peroxidases and Pseudoperoxidases

Many copper and iron complexes can be reduced by O^-₂ as well as by H₂O₂. According to the rates of reduction and the concentration of O^-₂ and H₂O₂, the metal complexes may serve either as catalyst of O^-₂ dismutation or as catalysts of the reaction between O^-₂ and H₂O₂ to form OH' radical (Haber-Weiss reaction). Various factors which influence whether metal complexes protect the biological systems from superoxide toxicity or enhance it are discussed.     

Effects of scavengers of oxygen free radicals on the anaerobic oxidation of 6-hydroxydopamine by H2O2

H₂O₂, a product of the aerobic autoxidation of 6-hydroxydopamine, is also consumed as a reactant, contributing progressively more to the oxidation as the concentration of O₂ becomes limiting H₂O₂ is a less effective oxidant than O₂, since the anaerobic peroxidatic oxidation of 6-hydrodopamine is slower than the aerobic oxidation by three orders of magnitude. The anaerobic peroxidation was inhibited by the hydroxyl scavengers mannitol (13–40%), glucose (41–62%) and benzoate (15–100%), implying a catalytic role for ^.OH. -9e strongly inhibitory action of desferrioxamine (76–91%), regardless of which other scavengers were present, suggests a specific role for iron in the reaction, despite the use of Chelex 100-treated buffers. Further addition of diethylenetriaminepentaacetate (DTPA), benzoate or formate to the desferrioxamine-treated reactions resulted in complete inhibition. In contrast, the presence of DTPA alone, accelerated the reaction by 160%. This acceleration is in part due to stimulation by DTPA of production of ^.OH (by Fenton-type reactions), since it was partially prevented by the hydroxyl scavengers benzoate (32% inhibition) and glucose (41%). Thus, DTPA inhibits the participation of metals other than iron, but potentiates the catalytic role of iron, in the reduction of hydrogen peroxide. The semidehydromannitol radical can reduce the DTPA-Fe³⁺ chelate directly, since mannitol further accelerated the DTPA-stimulated peroxidation (by 55%). Superoxide dismutase also accelerated the reaction (by 57–84%). This activation was seen regardless of which other scavengers were present. These effects are explained in terms of potentiating or moderating interactions among the reactive intermediates which propagate the overall reaction.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Non-invasive Analysis of Reactive Oxygen Species Generated in NH4OH-induced Gastric Lesions of Rats using a 300 MHz In Vivo ESR Technique

Free radicals are reportedly involved in mucosal injury, including NH⁴OH-induced gastric lesions, but the kind, location and origin of radical generation have yet to be clarified. We developed the non-invasive measurement of reactive oxygen species (ROS) in stomach, and applied to mucosal injury. NH⁴OH-induced gastric lesions were prepared in rats, which were then given a nitroxyl probe intragastrically or intravenously, and the spectra of the gastric region were obtained by in vivo 300?MHz electron spin resonance (ESR) spectroscopy. The spectral change of the nitroxyl probe administered intragastrically was significantly enhanced 30?min after NH⁴OH administration, but no change occurred when the probe was given by intravenous injection. The enhanced change was confirmed to be due to ?OH generation, because it was completely suppressed by mannitol, catalase and desferrioxamine (DFO), and was not observed in neutropenic rats. NH⁴OH-induced neutrophil infiltration of the gastric mucosa was suppressed by intravenous injection of superoxide dismutase (SOD) or catalase, or by administration of allopurinol. The present study provided the direct evidence in NH⁴OH-treated living rats that ?OH produced from O₂^?- derived from neutrophils caused gastric lesion formation, while O₂^?- or H²O² derived from the xanthine oxidase system in endothelial cells was involved in neutrophil infiltration.     

The role of interactions between O2, H2O2, .OH,e- and O2- in free radical damage to biological systems

The occurrence of the Haber-Weiss reaction and other interactions between free radicals has been investigated in the effects of mixtures of free radicals on the permeability of resealed erythrocyte ghosts and on the activity of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. The following mixtures were found to induce damage greater than that which could be accounted for by the independent actions of the constituent free radicals: (i) · OH + H₂O₂, and (ii) · OH + H₂O₂ + O₂^?. In contrast, the following mixtures were found to induce less damage than that predicted on the basis of independent actions of constituent free radicals: (i) H₂O₂ + O₂^?, and (ii) oxidizing radicals ( · OH, H₂O₂) + reducing radicals (e^?, H · ). These results suggest a Haber-Weiss-like interaction between H₂O₂ and O₂^?and an interaction between H₂O₂ and · OH to produce a species more potent than either in causing increased permeability. The decrease in damage observed in the simultaneous presence of oxidizing and reducing radicals suggests an antagonistic effect by which each tends to moderate damage by the other. Inactivation of glyceraldehyde-3-phosphate dehydrogenase was found to be more sensitive to radiation than permeability by an order of magnitude, while permeability was more sensitive to the enhancement of damage by oxygen. Comparison of the effectiveness of free radical scavengers in inhibiting the increase in permeability caused by free radicals showed the following order of effectiveness, expressed in terms of percentage protection: formate (90%) > nitrogen (65%) > catalase (60%) > dismutase (32%), and with respect to enzymatic inactivation, nitrogen (100%) > formate (77%) > dismutase (48%) > catalase (44%). The relative rates observed anaerobically and aerobically in the presence and absence of the above scavengers suggest that (at least in the case of radiation damage to the membranes of erythrocyte ghost cells) the “oxygen effect” is due to the interaction of oxygen with e^? and H., producing O₂^? which aggravates damage under conditions which allow consequent Haber-Weiss-like reactions. The further increase in damage when oxygen concentration is raised yet higher is due to the interaction of oxygen with the sites of initial damage.     

Microsensor Measurements of Sulfate Reduction and Sulfide Oxidation in Compact Microbial Communities of Aerobic Biofilms

The microzonation of O₂ respiration, H₂S oxidation, and SO₄^2- reduction in aerobic trickling-filter biofilms was studied by measuring concentration profiles at high spatial resolution (25 to 100 μm) with microsensors for O₂, S^2-, and pH. Specific reaction rates were calculated from measured concentration profiles by using a simple one-dimensional diffusion reaction model. The importance of electron acceptor and electron donor availability for the microzonation of respiratory processes and their reaction rates was investigated. Oxygen respiration was found in the upper 0.2 to 0.4 mm of the biofilm, whereas sulfate reduction occurred in deeper, anoxic parts of the biofilm. Sulfate reduction accounted for up to 50% of the total mineralization of organic carbon in the biofilms. All H₂S produced from sulfate reduction was reoxidized by O₂ in a narrow reaction zone, and no H₂S escaped to the overlying water. Turnover times of H₂S and O₂ in the reaction zone were only a few seconds owing to rapid bacterial H₂S oxidation. Anaerobic H₂S oxidation with NO₃^- could be induced by addition of nitrate to the medium. Total sulfate reduction rates increased when the availability of SO₄^2- or organic substrate increased as a result of deepening of the sulfate reduction zone or an increase in the sulfate reduction intensity, respectively.     

Multiple reactions in vanadyl-V(IV) oxidation by H2O2

Oxidation of vanadyl sulfate by H₂O₂ involves multiple reactions at neutral pH conditions. The primary reaction was found to be oxidation of V(IV) to V(V) using 0.5 equivalent of H₂O₂, based on the loss of blue color and the visible spectrum. The loss of V(IV) and formation V(V) compounds were confirmed by ESR and⁵¹V-NMR spectra, respectively. In the presence of excess H₂O₂ (more than two equivalents), the V(V) was converted into diperoxovanadate, the major end-product of these reactions, identified by changes in absorbance in ultraviolet region and by the specific chemical shift in NMR spectrum. The stoichiometric studies on the H₂O₂ consumed in this reaction support the occurrence of reactions of two-electron oxidation followed by complexing two molecules of H₂O₂. Addition of a variety of compounds—Tris, ethanol, mannitol, benzoate, formate (hydroxyl radical quenching), histidine, imidazole (singlet oxygen quenching), and citrate—stimulated a secondary reaction of oxygen-consumption that also used V(IV) as the reducing source. This reaction requires concomitant oxidation of vanadyl by H₂O₂, favoured at low H₂O₂:V(IV) ratio. Another secondary reaction of oxygen release was found to occur during vanadyl oxidation by H₂O₂ in acidic medium in which the end-product was not diperoxovanadate but appears to be a mixture of VO ₃ ⁺ (–546 ppm), VO³⁺ (–531 ppm) and VO ₂ ⁺ (–512 ppm), as shown by the⁵¹V-NMR spectrum. This reaction also occurred in phosphate-buffered medium but only on second addition of vanadyl. The compounds that stimulated the oxygen-consumption reaction were found to inhibit the oxygen-release reaction. A combination of these reactions occur depending on the proportion of the reactants (vanadyl and H₂O₂), the pH of the medium and the presence of some compounds that affect the secondary reactions.     

Acceptor side photoinhibition in PS II: On the possible effects of the functional integrity of the PS II donor side on photoinhibition of stable charge separation

Photoinhibition under aerobic and anaerobic conditions was analyzed in O₂-evolving and in Tris-treated PS II-membrane fragments from spinach by measuring laser-flash-induced absorption changes at 826 nm reflecting the transient P680⁺ formation and the chlorophyll fluorescence lifetime. It was found that anaerobic photoinhibitory treatment leads in both types of samples to the appearence of two long-lived fluorescence components with lifetimes of 7 ns and 16 ns, respectively. The extent of these fluorescence kinetics depends on the state of the reaction center (open/closed) during the fluorescence measurements: it is drastically higher in the closed state. It is concluded that this long-lived fluorescence is mainly emitted from modified reaction centers with singly reduced Q_A(Q_A ^-). This suggests that the observation of long-lived fluorescence components cannot necessarily be taken as an indicator for reaction centers with missing or doubly reduced and protonated Q_A (Q_AH₂). Time-resolved measurements of 826 nm absorption changes show that the rate of photoinhibition of the stable charge separation (P680^*Q_A P680⁺Q_A ^-), is nearly the same in O₂-evolving and in Tris-treated PS II-membrane fragments. This finding is difficult to understand within the framework of the Q_AH₂-mechanism for photoinhibition of stable charge separation because in that case the rate of photoinhibition should strongly depend on the functional integrity of the donor side of PS II. Based on the results of this study it is inferred, that several processes contribute to photoinhibition within the PS II reaction center and that a mechanism which comprises double reduction and protonation of Q_A leading to Q_AH₂ formation is only of marginal – if any – relevance for photoinhibition of PS II under both, aerobic and anaerobic, conditions.     

Hydrogen peroxide production by Zymomonas mobilis

Summary The anaerobic aerotolerant bacterium Zymomonas mobilis 113 produced superoxide (O ₂ ^- ) and hydrogen peroxide (H₂O₂) under aerobic conditions. The main generators of H₂O₂ were glucose oxidase and superoxide dismutase (SOD). The O ₂ ^- generation was probably related to minor alternative reduced nicotinamide adenine zinucleotide (NADH)-oxidation reactions in the electron transport chain. An increase in medium pO₂ was observed during growth of Z. mobilis 113 in a batch culture. The maximum pO₂ increase correlated with glucose oxidase and SOD activities. An decrease in medium pO₂ value coincided with an increase in catalase activity in batch culture. Medium deoxygenation reduced the pO₂ effect, yet the culture still responded with a pO₂ increase after inoculation and addition of the feeding medium. We conclude that the apparent pO₂ effects are related to changes in H₂O₂ concentration in the culture liquid.     

Effect of fungal co-cultures on ligninolytic enzyme activities,H2O2 production,and orange G discoloration

Abstract

In this study, the effects of Aspergillus niger in coculture with the basidiomycetes, Trametes versicolor, T. maxima, and Ganoderma spp., were studied to assess H₂O₂ production and laccase (Lac), Lignin Peroxidase (LiP), and manganese peroxidase (MnP) activities. The results indicated that maximum discoloration was of 97%, in the T. maxima and A. niger coculture, where the concentration of H₂O₂ was 5?mg/L and 6.3?mg/L in cultures without and with dye, respectively. These concentrations of H₂O₂ were 1.6- and 1.8-fold higher than monocultures of T. maxima (3.37?mg/L) and A. niger (3.87?mg/L), respectively. In the same coculture, the LiP and MnP enzyme activities also increased 12-fold, (from 0.08?U/mg to 0.99?U/mg), and 67-fold, (from 0.11?U/mg to 7.4?U/mg), respectively. The Lac activity increased 1.7-fold (from 13.46?U/mg to 24?U/mg). Further, a Box–Behnken experimental design indicated a 1.8-fold increase of MnP activity (from 7.4?U/mg to 13.3?U/mg). In addition, dye discoloration regression model obtained from the Box–Behnken experimental design showed a positively correlation with H₂O₂, (R²?=?0.58) and a negatively correlation with Lac activity (R² = –0.7).     

Graphene‐amplified electrogenerated chemiluminescence of CdTe quantum dots for H2O2 sensing

Electrogenerated chemiluminescence (ECL) of thiol‐capped CdTe quantum dots (QDs) in aqueous solution was greatly enhanced by PDDA‐protected graphene (P‐GR) film that were used for the sensitive detection of H₂O₂. When the potential was cycled between 0 and ?2.3 V, two ECL peaks were observed at ?1.1 (ECL‐1) and ?1.4 V (ECL‐2) in pH 11.0, 0.1 M phosphate buffer solution (PBS), respectively. The electron‐transfer reaction between individual electrochemically‐reduced CdTe nanocrystal species and oxidant coreactants (H₂O₂ or reduced dissolved oxygen) led to the production of ECL‐1. While mass nanocrystals packed densely in the film were reduced electrochemically, assembly of reduced nanocrystal species reacted with coreactants to produce an ECL‐2 signal. ECL‐1 showed higher sensitivity for the detection of H₂O₂ concentrations than that of ECL‐2. Further, P‐GR film not only enhanced ECL intensity of CdTe QDs but also decreased its onset potential. Thus, a novel CdTe QDs ECL sensor was developed for sensing H₂O₂. Light intensity was linearly proportional to the concentration of H₂O₂ between 1.0 × 10^?5 and 2.0 x 10^‐7 mol L^?1 with a detection limit of 9.8 x 10^?8 mol L^?1. The P‐GR thin‐film modified glassy carbon electrode (GCE) displayed acceptable reproducibility and long‐term stability. Copyright © 2012 John Wiley & Sons, Ltd.     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

Effects of oxygen on the dark recombination between photoreduced secondary quinone and oxidized bacteriochlorophyll in Rhodobacter sphaeroides reaction centers

The influence of duration of exposure to actinic light (from 1 sec to 10 min) and temperature (from 3 to 35^°C) on the temporary stabilization of the photomobilized electron in the secondary quinone acceptor (Q_B) locus of Rhodobacter sphaeroides reaction centers (RC) was studied under aerobic or anaerobic conditions. Optical spectrophotometry and ESR methods were used. The stabilization time increased significantly upon increasing the exposure duration under aerobic conditions. The stabilization time decreased under anaerobic conditions, its dependence on light exposure duration being significantly less pronounced. Generation of superoxide radical in photoactivated aerobic samples was revealed by the ESR method. Possible interpretation of the effects is suggested in terms of interaction between the semiquinone Q_B with oxygen, the interaction efficiency being determined by the conformational transitions in the structure of RC triggered by actinic light on and off.     

Methanotroph and methanogen coupling in granular biofilm under O2-limited conditions

Summary The co-culture between Methylosinus sporium, a strictly aerobic methanotroph, and strictly anaerobic methanogens was studied in 5 L aerobic/anaerobic coupled granular sludge reactors under O₂-limited conditions. The methanogenic bacteria maintained very good metabolic activities and were able to produce sufficient methane which serviced as substrate for methanotrophic growth. Although other strictly aerobic population proliferated by two orders of magnitude after the granular sludge had been operated under O₂-limited conditions for one month, only a limited amount of the added methanotroph remained in the sludge. This result may indicate that M. sporium lacks sufficient O₂ affinity to compete with facultative bacteria for the dissolved O₂ for their growth.     

Arbuscular mycorrhizae improve low temperature tolerance in cucumber via alterations in H2O2 accumulation and ATPase activity

The combined effects of arbuscular mycorrhizal fungi (AMF) and low temperature (LT) on cucumber plants were investigated with respect to biomass production, H₂O₂ accumulation, NADPH oxidase, ATPase activity and related gene expression. Mycorrhizal colonization ratio was gradually increased after AMF-inoculation. However, LT significantly decreased mycorrhizal colonization ability and mycorrhizal dependency. Regardless of temperature, the total fresh and dry mass, and root activity of AMF-inoculated plants were significantly higher than that of the non-AMF control. The H₂O₂ accumulation in AMF-inoculated roots was decreased by 42.44 % compared with the control under LT. H₂O₂ predominantly accumulated on the cell walls of apoplast but was hardly detectable in the cytosol or organelles of roots. Again, NADPH oxidase activity involved in H₂O₂ production was significantly reduced by AMF inoculation under LT. AMF-inoculation remarkably increased the activities of P-type H⁺-ATPase, P-Ca²⁺-ATPase, V-type H⁺-ATPase, total ATPase activity, ATP concentration and plasma membrane protein content in the roots under LT. Additionally, ATP concentration and expression of plasma membrane ATPase genes were increased by AMF-inoculation. These results indicate that NADPH oxidase and ATPase might play an important role in AMF-mediated tolerance to chilling stress, thereby maintaining a lower H₂O₂ accumulation in the roots of cucumber.     

The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR

In this study, the functions of two established Fe-S cluster biogenesis pathways, Isc (iron-sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc ⁻ strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O₂-stable [4Fe-4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by ⁵⁵Fe-labeling experiments. Using [³⁵S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O₂-inactivated apo-FNR into [4Fe-4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.     

Growth of Rhodopseudomonas palustris under phototrophic and non-phototrophic conditions and its CO-dependent H2 production

A new hydrogen producing bacterium, Rhodopseudomonas palustris P4, originally isolated under an anaerobic/phototrophic condition, grew well under aerobic/chemoheterotrophic or anaerobic/chemoheterotrophic conditions and showed CO-dependent, H₂ production activity when transferred to anaerobic conditions. Cell growth was best under an aerobic/chemoheterotrophic condition as the doubling time of 1 h, while the H₂ production activity was highest in the cells grown under an aerobic/chemoheterotrophic condition at 20 mmol g^–1 cell^–1 h^–1.     

Mechanism of Hydroxyl Radical Scavenging by Rebamipide: Identification of Mono-hydroxylated Rebamipide as a Major Reaction Product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (^?OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against ^?OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H²O²), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a ^?OH-trapping agent after UVB exposure (305?nm) to H²O² for 1?min in the presence of rebamipide. The signal intensity of ^?OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62?×?10¹⁰, 8.16?×?10⁹ and 1.65?×?10¹⁰?M^-1?s^-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the ^?OH generated by UVB/H²O². Specific formation of this product explained the molecular characteristics of rebamipide as a potential ^?OH scavenger.