DMPO Concentrations Research Articles

Estimation of the Local Concentration of the Markedly Dense Hydroxyl Radical Generation Induced by X-rays in Water.

The linear-density (number of molecules on an arbitrary distance) of X-ray-induced markedly dense hydroxyl radicals (•OH) in water was estimated based on EPR spin-trapping measurement. A lower (0.13 mM–2.3 M) concentration series of DMPO water solutions and higher (1.7–6.0 M) concentration series of DMPO water solutions plus neat DMPO liquid (8.8 M as DMPO) were irradiated with 32 Gy of X-rays. Then, the yield of DMPO-OH in DMPO water solutions and the total spin-adduct of DMPO in neat DMPO were quantified. For the higher concentration DMPO series, the EPR peak area was estimated by double integration, and the baseline correction of the integral spectrum is necessary for accurate estimation of the peak area. The preparation of a suitable standard sample corresponding to the electric permittivity according to DMPO concentration was quite important for quantification of DMPO-OH, especially in DMPO concentration beyond 2 M. The linear-density of •OH generation in water by X-ray irradiation was estimated from the inflection point on the plot of the DMPO-OH yield versus DMPO linear-density. The linear-density of X-ray-induced markedly dense •OH was estimated as 1168 μm−1, which was converted to 0.86 nm as the intermolecular distance and 2.6 M as the local concentration.

Effects of loading a magnetic field longitudinal to the linear particle-beam track on yields of reactive oxygen species in water

The effects of a magnetic field longitudinal to the ion beam track on the generation of hydroxyl radicals (•OH) and hydrogen peroxide (H2O2) in water were investigated. A longitudinal magnetic field was reported to enhance the biological effects of the ion beam. However, the mechanism of the increased cell death by a longitudinal magnetic field has not been clarified. The local density of •OH generation was estimated by a method based on the EPR spin-trapping. A series of reaction mixtures containing varying concentrations (0.76‒2278 mM) of DMPO was irradiated by 16 Gy of carbon- or iron-ion beams at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, NIRS/QST, Chiba, Japan) with or without a longitudinal magnetic field (0.0, 0.3, or 0.6 T). The DMPO-OH yield in the sample solutions was measured by X-band EPR and plotted versus DMPO density. O2-dependent and O2-independent H2O2 yields were measured. An aliquot of ultra-pure water was irradiated by carbon-ion beams with or without a longitudinal magnetic field. Irradiation experiments were performed under air or hypoxic conditions. H2O2 generation in irradiated water samples was quantified by an EPR spin-trapping, which measures •OH synthesized from H2O2 by UVB irradiation. Relatively sparse •OH generation caused by particle beams in water were not affected by loading a magnetic field on the beam track. O2-dependent H2O2 generation decreased and oxygen-independent H2O2 generation increased after loading a magnetic field parallel to the beam track. Loading a magnetic field to the beam track made •OH generation denser or made dense •OH more reactive.

Stability of 5,5-dimethyl-1-pyrroline-N-oxide as a spin-trap for quantification of hydroxyl radicals in processes based on Fenton reaction

Fenton reaction was used to produce hydroxyl radicals under conditions similar to AOPs with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap agent in electron paramagnetic resonance (EPR) analysis. A theoretical kinetics model was developed to determine conditions under which the spin-adduct DMPO-OH is not further oxidized by Fe3+ and excessive radicals, so that hydroxyl radicals concentration could be accurately inferred. Experiments were designed based upon the model and H2O2 and Fe2+ concentrations were varied from 1 to 100 mM and from 0.1 to 10 mM, respectively, with a constant H2O2: Fe2+ ratio of 10:1. Results confirmed that DMPO concentration should be at least 20 times higher than the concentration of H2O2 and 200 times higher than iron concentration to produce stable DMPO-OH EPR signal. When DMPO: H2O2 ratio varied from 1 to 10, DMPO-OH could generate intermediates and be further oxidized leading to the apparition of an additional triplet. This signal was attributed to a paramagnetic dimer: its structure and a formation mechanism were proposed. Finally, the utilization of sodium sulfite and catalase to terminate Fenton reaction was discussed. Catalase appeared to be compatible with DMPO. However, sodium sulfite should be avoided since it reacted with DMPO-OH to form DMPO-SO3.

Density of hydroxyl radicals generated in an aqueous solution by irradiating carbon-ion beam.

The density of hydroxyl radicals (·OH) produced in aqueous samples by exposure to X-ray or carbon-ion beams was investigated. The generation of ·OH was detected by the electron paramagnetic resonance (EPR) spin-trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping agent. When the concentration of DMPO is in excess of the generated ·OH, the production of DMPO-OH (spin-trapped ·OH) should be saturated. Reaction mixtures containing several concentrations (0.5-1685 mM) of DMPO were then irradiated by a 32 Gy 290 MeV carbon-ion beam (C290-beam) or X-ray. C290-beam irradiation was performed at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, National Institute of Radiological Sciences, Chiba, Japan), applying different linear energy transfers (LET) (20-169 keV/µm). The amount of DMPO-OH in the irradiated samples was detected by EPR spectroscopy. The generation of DMPO-OH increased with the concentration of initial DMPO, displayed a shoulder around 3.3 mM DMPO, and reached a plateau. This plateau suggests that the generated ·OH were completely trapped. Another linear increase in DMPO-OH measured in solutions with higher DMPO concentrations suggested very dense ·OH generation (>1.7 M). Generation of ·OH is expected to be localized on the track of the radiation beam, because the maximum concentration of measured DMPO-OH was 40 µM. These results suggested that both sparse (≈3.3 mM) and dense (>1.7 M) ·OH generation occurred in the irradiated samples. The percentage of dense ·OH generation increased with increasing LET. Different types of dense ·OH generation may be expected for X-ray and C290-beams.

Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase

Low-density lipoprotein (LDL) oxidation is the primary event in atherosclerosis, and LDL lipoperoxidation leads to modifications in apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno-spin trapping, we detected the formation of protein free radicals on LDL incubated with Cu2+ or the soybean lipoxidase (LPOx)/phospholipase A2 (PLA2). With low concentrations of DMPO (1mM), Cu2+ dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu2+ showed maximum yields after 30min. In contrast, the yields of apo B-100 radicals formed by LPOx/PLA2 followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate, and Trolox dose-dependently reduced apo B-100 free radical formation in LDL exposed to Cu2+. In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA2. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu2+ or LPOx/PLA2 for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu2+, when we used the oxidizing system LPOx/PLA2, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans.

EPR study of 17O-enriched titania nanopowders under UV irradiation

Anatase and rutile nanoparticulate samples, prepared via TiCl4 hydrolysis using either ordinary or 17O-enriched water (up to 90%) with subsequent heat-treatment under vacuum, were investigated by EPR spectroscopy upon UV photoexcitation in the temperature range 100–160K. The experimental EPR spectra of photoexcited anatase Ti16O2 revealed the presence of paramagnetic signals attributed to Ti(III) species, two kinds of O− centers produced from lattice O2− ions, along with O2−/HO2 radicals which originated from adsorbed molecular oxygen. The presence of 17O atoms in the anatase crystal lattice was reflected in substantial EPR line broadening and the experimental spectrum was interpreted as a superposition of signals attributed to Ti(III) with axial symmetry arising from an interaction with an 17O nucleus (g⊥=1.962, g||=1.923; A⊥=1.68mT, A||=1.61mT), the signal of a photogenerated hole 17O− (g⊥=2.019, g||=2.004; A⊥=1.76mT, A||=10.61mT) and O2−. Due to the thermal treatment at higher temperature (1000°C) both rutile samples showed a variety of paramagnetic signals attributed to Ti(III) sites already present before photoexcitation. The EPR spectrum of 17O-enriched rutile sample was simulated as a superposition of Ti(III)–17O sites and photogenerated ozonide in the rutile matrix. The ability of synthesized anatase and rutile nanopowders to produce hydroxyl radicals upon UV photoexcitation in the aerated aqueous suspensions was monitored via an EPR spin trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent. Upon continuous exposure of titania suspensions a typical four-line EPR signal of the DMPO–OH spin-adduct was found (AN=1.492mT, AH=1.476mT; g=2.0059). The dependence of the DMPO–OH concentration on the irradiation time was similar for 16O- and 17O-anatase samples, as well as for both rutile samples. The concentration of DMPO–OH spin-adduct, after 10-min exposure, monitored for rutile was reduced by approximately 40% in comparison with anatase.

Environmentally Persistent Free Radicals (EPFRs)-2. Are Free Hydroxyl Radicals Generated in Aqueous Solutions?

A chemical spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), in conjunction with electron paramagnetic resonance (EPR) spectroscopy was employed to measure the production of hydroxyl radical (·OH) in aqueous suspensions of 5% Cu(II)O/silica (3.9% Cu) particles containing environmentally persistent free radicals (EPFRs) of 2-monochlorophenol (2-MCP). The results indicate: (1) a significant differences in accumulated DMPO-OH adducts between EPFR containing particles and non-EPFR control samples, (2) a strong correlation between the concentration of DMPO-OH adducts and EPFRs per gram of particles, and (3) a slow, constant growth of DMPO-OH concentration over a period of days in solution containing 50 μg/mL EPFRs particles + DMPO (150 mM) + reagent balanced by 200 μL phosphate buffered (pH = 7.4) saline. However, failure to form secondary radicals using standard scavengers, such as ethanol, dimethylsulfoxide, sodium formate, and sodium azide, suggests free hydroxyl radicals may not have been generated in solution. This suggests surface-bound, rather than free, hydroxyl radicals were generated by a surface catalyzed-redox cycle involving both the EPFRs and Cu(II)O. Toxicological studies clearly indicate these bound free radicals promote various types of cardiovascular and pulmonary disease normally attributed to unbound free radicals; however, the exact chemical mechanism deserves further study in light of the implication of formation of bound, rather than free, hydroxyl radicals.

Scavenging of superoxide anion radical and hydroxyl radical by novel thiazolyl‐thiazolidine‐2,4‐dione compounds

The antioxidant behavior of a series of new synthesized substituted thiazolyl-thiazolidine-2,4-dione compounds (TZDs) was examined using chemiluminescence and electron paramagnetic resonance spin trapping techniques. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used as the spin trap. The reactivity of TZDs with superoxide anion radical (O2(*-)) and hydroxyl radical (HO(*)) was evaluated using potassium superoxide/18-crown-6 ether dissolved in dimethylsulfoxide, and the Fenton-like reaction (Fe(2+) + H2O2), respectively. The results showed that TZDs efficiently inhibited light emission from the O2(*-) generating system at a concentration of 0.05-1 mmol L(-1) (5-94% reductions were found at 1 mmol L(-1) concentration). The TZD compounds showed inhibition of HO(*)-dependent DMPO-OH spin adduct formation from DMPO (the amplitude decrease ranged from 8 to 82% at 1 mmol L(-1) concentration). The findings showed that examined TZDs had effective activities as radical scavengers.

The Radical Trap 5,5-Dimethyl-1-PyrrolineN-Oxide Exerts Dose-Dependent Protection against Myocardial Ischemia-Reperfusion Injury through Preservation of Mitochondrial Electron Transport

Free radicals are important mediators of myocardial ischemia-reperfusion injury. Nitrone spin traps have been shown to scavenge free radicals. The cardioprotective effect of the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was investigated in an isolated heart model of global ischemia and reperfusion. Rat hearts were perfused and subjected to global ischemia for 30 min followed by reperfusion with four treatment groups of varying DMPO concentration (0.5-10 mM) administered before induction of ischemia. DMPO treatment improved the recovery of left ventricular (LV) function and coronary flow over the 30-min period of reperfusion compared with untreated hearts. Enhanced recovery was observed for all doses studied but was highest with 1 mM treatment with 2.4-fold higher recovery of LV developed pressure and 37% reduction in infarct size. Superoxide was measured by tissue fluorometry using the O(2)* probe hydroethidine. Hearts treated with 1 mM DMPO showed a significant reduction in O(2)* production compared with control hearts both over the first 5 min of ischemia and upon reperfusion after 30 min of global ischemia. Studies of mitochondrial function demonstrated that 1 mM DMPO increased the recovery of function of complexes I, II/III, and IV after 30 min of reperfusion. Immunoblotting with antibodies against complexes I, II, and IV further revealed marked up-regulation of mitochondrial proteins, suggesting that DMPO prevents their ischemic degradation via scavenging oxygen radicals generated during ischemia/reperfusion. Thus, DMPO functions as a protective agent against ischemic and postischemic injury via radical scavenging, conferring robust dose-dependent protection with salvage of mitochondrial function and redox homeostasis.

Antioxidant Activity of Caffeic Acid through a Novel Mechanism under UVA Irradiation

Effect of caffeic acid on the formation of hydroxyl radicals was examined during xanthone-mediated photosensitization. The reaction was performed on irradiation (λ = 365 nm) of the standard reaction mixture containing 15 µM xanthone, 0.1 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 20 mM phosphate buffer (pH 7.4) using electron paramagnetic resonance (EPR) with spin trapping. Caffeic acid inhibited the formation of hydroxyl radicals. Caffeic acid hardly scavenged both hydroxyl radicals and superoxide radicals under conditions employed in this paper in spite of its ability to act as a hydrogen donor or a reagent for the aromatic hydroxylation, because high concentration of DMPO trapped hydroxyl radicals overwhelmingly. Furthermore, caffeic acid inhibited the formation of hydroxyl radicals in the standard reaction mixture with EDTA under UVA irradiation. Accordingly, the inhibitory effect of caffeic acid on the formation of hydroxyl radicals in the standard reaction mixture under UVA irradiation is not due to its ability to chelate iron. Thus, the inhibitory effect of caffeic acid seems to occur in the standard reaction mixture under UVA irradiation through a novel antioxidation activity, i.e., ability to quench the exited xanthone.

Spin Trapping Investigation of Peroxide- and Isoniazid-Induced Radicals in Mycobacterium tuberculosis Catalase-Peroxidase

A new approach, the immuno-spin trapping assay, used a novel rabbit polyclonal anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antiserum to detect protein radical-derived DMPO nitrone adducts in the hemoprotein Mycobacterium tuberculosis catalase-peroxidase (KatG). This work demonstrates that the formation of protein nitrone adducts is dependent on the concentrations of tert-BuOOH and DMPO as shown by Western blotting and an enzyme-linked immunosorbent assay (ELISA). We have also detected protein-protein cross-links formed during turnover of Mtb KatG driven by tert-butyl peroxide ( tert-BuOOH) or enzymatic generation of hydrogen peroxide. DMPO inhibits this dimerization due to its ability to trap the amino acid radicals responsible for the cross-linkage. Chemical modifications by tyrosine and tryptophan blockage suggest that tyrosine residues are one site of formation of the dimers. The presence of the tuberculosis drug isoniazid (INH) also prevented cross-linking as a result of its competition for the protein radical. Protein-DMPO nitrone adducts were also generated by a continuous flux of hydrogen peroxide. These findings demonstrated that the protein-based radicals were formed not only during Mtb KatG turnover with alkyl peroxides but also in the presence of hydrogen peroxide. Furthermore, the formation of protein-DMPO nitrone adducts was accelerated in the presence of isoniazid.

Electron Transfer between a Tyrosyl Radical and a Cysteine Residue in Hemoproteins: Spin Trapping Analysis

We investigated electron transfer between a tyrosyl radical and cysteine residue in two systems, oxyhemoglobin (oxyHb)/peroxynitrite/5,5-dimethyl-1-pyrroline N-oxide (DMPO) and myoglobin (Mb)/hydrogen peroxide/DMPO, using a combination of techniques including ESR, immuno-spin trapping (IST), and ESI/MS. These techniques show that the nitrone spin trap DMPO covalently binds to one or more amino acid radicals in the protein. Treating oxyHb with peroxynitrite and Mb with H2O2 in the presence of a low DMPO concentration yielded secondary Cys-DMPO radical adduct exclusively, whereas in the presence of high DMPO, more of the primary Tyr-DMPO radical adduct was detected. In both systems studied, we found that, at high DMPO concentrations, mainly tyrosyl radicals (Hb-Tyr42/Tyr24 and Mb-Tyr103) are trapped and the secondary electron-transfer reaction does not compete, whereas in the presence of low concentrations of DMPO, the secondary reaction predominates over tyrosyl trapping, and a thiyl radical is formed and then trapped (Hb-Cys93 or Mb-Cys110). With increasing concentrations of DMPO in the reaction medium, primary radicals have an increasing probability of being trapped. MS/MS was used to identify the specific Tyr and Cys residues forming radicals in the myoglobin system. All data obtained from this combination of approaches support the conclusion that the initial site of radical formation is a Tyr, which then abstracts an electron from a cysteine residue to produce a cysteinyl radical. This complex phenomenon of electron transfer from one radical to another has been investigated in proteins by IST, ESR, and MS.

Effects of Hydrodynamic Conditions on OH Radical Production at Ti/Pt Anodes During Electrochemical Treatment

The effects of hydrodynamic conditions on the production of hydroxyl radicals (•OH) during electrochemical treatment using a titanium coated by platinum anode and a stainless steel cathode are discussed in this paper. The sample used was ultra-pure water containing 200 mM of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The amount of •OH for treatment was measured using electron spin resonance spectrometry coupled with DMPO spin trapping. Under constant hydrodynamic conditions, the production rate of the •OH spin adduct (DMPO-OH) increased with the terminal potential. This result was caused by the change in the electric current density at the anode, which was proportional to the rate of production of DMPO-OH. Increasing the linear velocity of water in an electrolytic cell promoted the production of DMPO-OH by two effects, namely, by the enhancement of ion transportation and the promotion of DMPO transfer from the bulk to the anode. The former effect emerged when the DMPO concentration near the anode was not insignificant in comparison with the DMPO concentration in the bulk, that is, when the overall rate-determining step was the electron transfer at the anode. The latter effect emerged when the DMPO concentration near the anode was much lower than the DMPO concentration in the bulk, that is, when the overall rate-determining step was the diffusion of DMPO from the bulk to the anode. In addition, the latter effect was found to be proportional to the square root of the linear velocity of water in the electrolytic cell.

Nitric oxide release from the unimolecular decomposition of the superoxide radical anion adduct of cyclic nitrones in aqueous medium

Nitrones such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO) have become the spin-traps of choice for the detection of transient radical species in chemical and biological systems using electron paramagnetic resonance (EPR) spectroscopy. The mechanism of decomposition of the superoxide radical anion (O2(.-)) adducts of DMPO, DEPMPO and EMPO in aqueous solutions was investigated. Our findings suggest that nitric oxide (NO) was formed during the decomposition of the O2(.-) adduct as detected by EPR spin trapping using Fe(II)N-methyl-d-glucamine dithiocarbamate (MGD). Nitric oxide release was observed from the O2(.-) adduct formed from hypoxanthine-xanthine oxidase, PMA-activated human neutrophils, and DMSO solution of KO2. Nitric oxide formation was not observed from the independently generated hydroxyl radical adduct. Formation of nitric oxide was also indirectly detected as nitrite (NO2(.-)) utilizing the Griess assay. Nitrite concentration increases with increasing O2(.-) concentration at constant DMPO concentration, while NO2(.-) formation is suppressed at anaerobic conditions. Moreover, large excess of DMPO also inhibits NO2(.-) formation which can be attributed to the oxidation of DMPO to hydroxamic acid nitroxide (DMPO-X) by nitrogen dioxide (NO2), a precursor to NO2(.-). Product analysis was also conducted to further elucidate the mechanism of adduct decay using gas chromatography-mass spectrometry (GC-MS) technique.

An experimental and theoretical model for solar UVA-irradiation of soluble eumelanin: towards modelling UVA-photoreactions in the melanosome?

A model is developed for the UVA-irradiation of soluble eumelanin exposed to levels of irradiation comparable to sunlight. Radical production was determined in soluble dl- and l-dopa melanins exposed to solar levels of UVA, using electron spin resonance spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Steady-state concentrations of DMPO–O 2H , which increased up to 0.3 mg/ml melanin, and then declined above 0.3 mg/ml, were detected at pH 4.5. The kinetic model incorporated the photosensitising and radical-scavenging reactions of eumelanin, and assumed semiquinone radical reduction of oxygen to be fast compared to disproportionation. The model is consistent with experimental data for melanin concentrations <0.1 mg/ml; but >0.1 mg/ml melanin is consistent only with data at raised oxygen tension. The rate-constant for reaction of the melanin semiquinone-radical and oxygen is estimated to be 10 3 mol −1 dm 3 s −1. In this model, where DMPO competes with melanin for HO 2 , at ambient oxygen levels, eumelanin exposed to solar levels of UVA photosensitises superoxide at concentrations <0.3 mg/ml melanin, and is increasingly stable towards oxidation when >0.3 mg/ml concentration. Eumelanin could have a negligible screening effect <0.1 mg/ml and very strong screening >1 mg/ml. This model would be biologically relevant if soluble forms of eumelanin were shown to exist in vivo, and is potentially useful for studies of the photochemistry and photophysics of eumelanin and phaeomelanin and to explore the effects of metal-ions, proteins and lipids in a model system.

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

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

Immunochemical detection of hemoglobin-derived radicals formed by reaction with hydrogen peroxide: involvement of a protein-tyrosyl radical.

To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/H2O2 system, we have used a new approach called "immuno-spin trapping," which combines the specificity and sensitivity of both spin trapping and antigen:antibody interactions. Previously, a novel rabbit polyclonal anti-DMPO nitrone adduct antiserum, which specifically recognizes protein radical-derived nitrone adducts, was developed and validated in our laboratory. In the present study, the formation of nitrone adducts on hemoglobin was shown to depend on the oxidation state of the iron heme, the concentrations of H2O2 and DMPO, and time as determined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The presence of reduced glutathione or L-ascorbate significantly decreased the level of nitrone adducts on metHb in a dose-dependent manner. To confirm the ELISA results, Western blotting analysis showed that only the complete system (oxy- or metHb/DMPO/H2O2) generates epitopes recognized by the antiserum. The specific modification of tyrosine residues on metHb by iodination nearly abolished antibody binding, while the thiylation of cysteine residues caused a small but reproducible decrease in the amount of nitrone adducts. These findings strongly suggest that tyrosine residues are the site of formation of the immunochemically detectable hemoglobin radical-derived nitrone adducts. In addition, we were able to demonstrate the presence of hemoglobin radical-derived nitrone adducts inside red blood cells exposed to H2O2 and DMPO. In conclusion, our new approach showed several advantages over EPR spin trapping with the anti-DMPO nitrone adduct antiserum by demonstrating the formation of tyrosyl radical-derived nitrone adduct(s) in human oxyHb/metHb at much lower concentrations than was possible with EPR and detecting radicals inside RBC exposed to H2O2.

Singlet oxygen-mediated hydroxyl radical production in the presence of phenols: whether DMPO-*OH formation really indicates production of *OH?

The reaction of singlet oxygen (1O2) generated by ultraviolet-A (UVA)-visible light (lambda > 330 nm) irradiation of air-saturated solutions of hematoporphyrin with phenolic compounds in the presence of a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), gave an electron spin resonance (ESR) spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-*OH). In contrast, the ESR signal of 5,5-dimethyl-2-pyrrolidone-N-oxyl, an oxidative product of DMPO, was observed in the absence of phenolic compounds. The ESR signal of DMPO-*OH decreased in the presence of either a *OH scavenger or a quencher of *O2 and under anaerobic conditions, whereas it increased depending on the concentration of DMPO. These results indicate both 1O2- and DMPO-mediated formation of free *OH during the reaction. When DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), no DEPMPO adduct of oxygen radical species was obtained. This suggests that 1O2, as an oxidizing agent, reacts little with DEPMPO, in which a strong electron-withdrawing phosphoryl group increases the oxidation potential of DEPMPO compared with DMPO. A linear correlation between the amounts of DMPO-*OH generated and the oxidation potentials of phenolic compounds was observed, suggesting that the electron-donating properties of phenolic compounds contribute to the appearance of *OH. These observations indicate that 1O2 reacts first with DMPO, and the resulting DMPO-1O2 intermediate is immediately decomposed/reduced to give *OH. Phenolic compounds would participate in this reaction as electron donors but would not contribute to the direct conversion of 1O2 to *OH. Furthermore, DEPMPO did not cause the spin-trapping agent-mediated generation of *OH like DMPO did.

Determination of antioxidant activity of herbs by ESR.

Water extracts of 32 herbs that are constituents of curry and curry powder were screened for superoxide anion radical (O2.-) scavenging activity. Among the screened samples, only clove, allspice, and basil were shown to decrease DMPO-O2.- adduct yields by more than 50% at 0.25 mg/mL as measured by an ESR spin trapping technique based on the HPX-XOD reaction. To study the mechanism of the O2.- scavenging activity, Km values were obtained from a Lineweaver-Burk plot for XOD in the presence of different concentrations of HPX, and the IC50 values at different DMPO concentrations were compared. Clove and basil directly eliminated O2.- like superoxide dismutase (SOD), whereas allspice reduced the amount of O2.- by inhibition of formation of O2.-.

Photobiological model studies on perester and pyridone tert-butoxyl radical sources (photo-Fenton-type reagents): 2'-deoxyguanosine modification by methyl radicals generated through competitive beta-cleavage in aqueous media.

In aqueous solution, UV irradiation of the photo-Fenton-type reagents perester 1 and N-tert-butoxypyridone 2 leads to the formation of tert-butoxyl radicals and methyl radicals as confirmed by product studies and by spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), followed by EPR spectroscopy. The methyl radicals result from the initially released tert-butoxyl radicals through beta-cleavage, a fragmentation pathway, which dominates at lower DMPO concentrations. In the presence of 2'-deoxyguanosine (dG), both photochemical radical sources afford 8-MedG [(2.3 +/- 0.3)%] and N7-MedG [(0.27 +/- 0.05)%] as modified products, whereas the direct reaction of the tert-butoxyl radicals with dG cannot compete with this beta-cleavage process.