The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution in chloroplasts.

The rate of reaction between superoxide anion (O2) and 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron) was measured with pulse radiolysis-generated O2. A kinetic spectrophotometric method utilizing competition between p-benzoquinone and tiron for O2 was employed. In this system, the known rate of reduction of p-benzoquinone was compared with the rate of oxidation of tiron to the semiquinone. From the concentration dependence of the rate of tiron oxidation, the absolute second order rate constant for the reaction was determined to be 5x10-8 M-minus1-s-minus1. Ascorbate reduced O2 to hydrogen peroxide with a rate constant of 10-8 M-minus1-s-minus1 as determined by the same method. The tiron semiquinone may be used as an indicator free radical for the formation of superoxide anion in biological systems because of the rapid rate of oxidation of the catechol by O2 compared to the rate of O2 formation is most enzymatic systems. Tiron oxidation was used to follow the formation of superoxide anion in swollen chloroplasts. The chloroplasts photochemically reduced molecular oxygen which was further reduced to hydrogen peroxide by tiron. Tiron oxidation specifically required O2 since O2 was consumed in the reaction and tiron did not reduce the P700 cation radical or other components of Photosystem I under anaerobic conditions.     

Pulse-radiolytic investigations of catechols and catecholamines II. Reactions of Tiron with oxygen radical species

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (°OH), superoxide anions (O₂^?) or a combination of both oxygen radicals. The rate constant with °OH radicals was determined at 1.0·10⁹ M^?1·s^?1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11–16), the rate constant with O₂^? of 1.0·10⁷ M^?1·s^?1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with °OH and O₂^? of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of °OH radicals, leading to the same products with catechol compounds.     

Oxidation of manganous pyrophosphate by superoxide radicals and illuminated spinach chloroplasts.

The oxidation of Mn²⁺-pyrophosphate to Mn³⁺ by superoxide (O₂^?) was quantitative as evidenced from the formation of Mn³⁺-pyrophosphate and hydrogen peroxide and from the inhibition by superoxide dismutase. Using the competitive relation between Mn²⁺-pyrophosphate and superoxide dismutase for the O₂^?, the rate constant of Mn²⁺ oxidation was estimated to be about 6 × 10⁶m^?1 s^?1. The oxidation of Mn²⁺-pyrophosphate by illuminated chloroplasts was also indicated to be stoichiometrically induced by O₂^?. In the presence of saturating amounts of the Mn²⁺, a double enhancement of hydrogen peroxide production and triple uptake of oxygen were found, as expected from the oxidation of Mn²⁺-pyrophosphate by O₂^?. Anaerobiosis or superoxide dismutase annuled these increments. We propose that the O₂^? generated as the sole initial step of the Mehler reaction oxidized Mn²⁺-pyrophosphate, and we discuss the role of free manganese in chloroplasts.     

The participation of a tryptophan residue in the binding of ferric iron to pyrocatechase

The fast reaction technique of pulse radiolysis was used to produce free radicals in aqueous solution from alcohols, deoxyribose, cytosine, uracil, thymine, dihydrothymine and histidine. The electron transfer reactions from these radicals to p-benzoquinone was observed from the formation kinetics of the semiquinone anion ·BQ^? at 430 nm and the efficiency was found to be as high as 90% or more, with k～5×10⁹ M^?1sec^?1. In acid or neutral solutions in the presence of oxygen the peroxy radicals ·O₂RH formed do not essentially transfer an electron to BQ, and the efficiency is <10%. The significance of these results in the fixation of radiation damage in photobiology and radiation biology are indicated. The reactions of the superoxide ·O₂^? radical with BQ are also presented and discussed.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

The allomerization of chlorophylls a and b with superoxide anion

The interaction of chlorophylls a and b with electrochemically prepared superoxide anion was studied in aprotic solvent. It was found that O₂^?·causes the deprotonation at carbon C-10 of ring V and production of chlorophyll enolate ions. The intermediate anions undergo rapid oxidation into corresponding chlorins. Pyrochlorophyll a, which lacks the C-10 carboxymethyl group, did not show the transformation. It is suggested that more strong free radical oxidants (e.g., HO₂·, or RO₂·) are capable of abstracting the hydrogen atom at C-10. The possible significance of free radical deprotonation and oxidation in chlorophyll allomerization is discussed.     

Nitric oxide donor-induced hyperpermeability of cultured intestinal epithelial monolayers: role of superoxide radical,hydroxyl radical,and peroxynitrite

Many of the cytopathic effects of nitric oxide (NO^·) are mediated by peroxynitrite (PN), a product of the reaction between NO^· and superoxide radical (O^·?₂). In the present study, we investigated the role of PN, O^·?₂ and hydroxyl radical (OH^·) as mediators of epithelial hyperpermeability induced by the NO^· donor, S-nitroso-N-acetylpenicillamine (SNAP), and the PN generator, 3-morpholinosydnonimine (SIN-1). Caco-2_BBe enterocytic monolayers were grown on permeable supports in bicameral chambers. Epithelial permeability, measured as the apical-to-basolateral flux of fluorescein disulfonic acid, increased after 24 h of incubation with 5.0 mM SNAP or SIN-1. Addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO^· scavenger, or Tiron, an O^·?₂ scavenger, reduced the increase in permeability induced by both donor compounds. The SNAP-induced increase in permeability was prevented by allopurinol, an inhibitor of xanthine oxidase (a source of endogenous O^·?₂). Diethyldithiocarbamate, a superoxide dismutase inhibitor, and pyrogallol, an O^·?₂ generator, potentiated the increase in permeability induced by SNAP. Addition of the PN scavengers deferoxamine, urate, or glutathione, or the OH^· scavenger mannitol, attenuated the increase in permeability induced by both SNAP and SIN-1. Both donor compounds decreased intracellular levels of glutathione and protein-bound sulfhydryl groups, suggesting the generation of a potent oxidant. These results support a role for PN, and possibly OH^·, in the pathogenesis of NO^· donor-induced intestinal epithelial hyperpermeability.     

Chemiluminescence investigations of antioxidative activities of some antibiotics against superoxide anion radical

A chemiluminescent technique was applied to determine antioxidative activities of adriamycin, farmorubicin, mitomycin C and bleomycin against superoxide anion radical (O₂^?) in aprotic medium. The antioxidant capacity was expressed as the decrease in light emission from the O₂^? solution by and antibiotic. A KO₂ solution in dimethyl sulphoxide (DMSO) and 18‐crown‐6 ether were used for the generation of O₂^?. The results showed that the examined compounds decreased the chemiluminescence (CL) sum from the O₂^?‐generating system in a dose‐dependent manner. Among the antibiotics examined, adriamycin, farmorubicin and bleomycin exhibited antioxidant activity almost comparable to that of 1,2‐dihydroxy benzene‐3,5‐disulphonic acid (tiron), an efficient of the O₂^? inhibitor. Mitomycin C was two‐times less effective as tiron in decreasing the initial CL intensity. The proposed assay with usage of ultraweak CL technique and the KO₂–DMSO–crown ether system was useful for the evaluation of antioxidant activity in aprotic solvents. Copyright © 2011 John Wiley & Sons, Ltd.     

Calcium-mediated enhancement of copper tolerance in Elodea canadensis

The alleviative effects of exogenous calcium on copper phytotoxicity were investigated in Elodea canadensis plants. There was a significant accumulation of Cu in the plants after their exposure to 0.01 mM Cu accompanied by many symptoms of toxicity. Increased uptake of Cu severely reduced content of photosynthetic pigments, soluble proteins, and free proline. The total antioxidant capacity (T-AOC), reduced glutathione (GSH), and non-protein thiol (NP-SH) were severely suppressed in Cu-stressed plants resulting in a rapid increase in content of superoxide anion (O₂ ^·?), hydrogen peroxide, lipid peroxidation, and cell death. Simultaneous application of Ca markedly increased the content of photosynthetic pigments, soluble proteins, free proline, T-AOC, GSH, and NP-SH, and reduced oxidative damage as indicated by lowered content of MDA, O₂ ^·?, and H₂O₂; and decreased cell death. Furthermore, application of Ca reduced Cu uptake and effectively reversed the Cu-induced nutrient imbalance.     

Active oxygen species in pea seedlings during the interactions with symbiotic and pathogenic microorganisms

The level of active oxygen species (AOS)—superoxide anion radical (O ₂ ^·? ) and hydrogen peroxide (H₂O₂)—in pea (Pisum sativum L.) cultivar Marat seedlings was studied upon their inoculation with symbiotic (Rhizobium leguminosarum bv. viceae strain CIAM 1026) and pathogenic (Pseudomonas syringae pv. pisi Sackett) microorganisms. Different patterns of the changes in AOS in pea seedlings during the interactions with the symbiont and the phytopathogen were recorded. It is assumed that O ₂ ^·? and H₂O₂ are involved in the defense and regulatory mechanisms of the host plant.     

Cobalt-induced oxidative stress causes growth inhibition associated with enhanced lipid peroxidation and activates antioxidant responses in Indian mustard (Brassica juncea L.) leaves

The effect of 100 μM cobalt (Co) on plant growth and on biochemical parameters indicative of oxidative stress was investigated in a hydroponic experiment. The responses of antioxidant enzymes and compounds of the ascorbate–glutathione (AsA–GSH) cycle were also assessed on the hyperaccumulating plant, Indian mustard (Brasssica juncea L.). The effect of excess Co was associated with a significant increase in the levels of proline, carbonylated protein, malondialdehyde, superoxide anion (O ₂ ^·? ), and hydrogen peroxide (H₂O₂), and resulted in the accumulation of Co. Co toxicity was associated with an increase in the volume of palisade and spongy cells, and a reduction in the number of chloroplasts per cell. Co-induced cell death was characterized by DNA fragmentation and a 36 kDa DNase activity. Despite decreased catalase activity, peroxidase, superoxide dismutase, and AsA–GSH cycle-related enzymes including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase exhibited remarkable induction under Co stress. Furthermore, the contents of reduced and oxidized forms of ascorbate and glutathione were significantly increased with Co supplementation. Co treatment led to the activation of 44 and 46 kDa mitogen-activated protein kinase (MAPK) and indicated the role of the MAPK cascade in transducing Co-mediated signals. The present results suggest that excess Co reduces seedling growth by inducing oxidative stress related to lipid peroxidation and overproduction of O ₂ ^·? and H₂O₂. The stimulated activities of antioxidative enzymes and induction of MAPKs did not reverse the oxidative stress caused by Co-induced reactive oxygen species generation in Indian mustard seedlings.     

Control of superoxide and nitric oxide formation during human sperm capacitation

We studied the modulation of superoxide anion (O₂·^?) and nitric oxide (NO·) generation during human sperm capacitation (changes needed for the acquisition of fertility). The production of NO· (diaminofluorescein-2 fluorescence assay), but not that of O₂·^? (luminescence assay), related to sperm capacitation was blocked by inhibitors of protein kinase C, Akt, protein tyrosine kinase, etc., but not by those of protein kinase A. Extracellular calcium (Ca²⁺) controlled O₂·^? synthesis but extra- and intracellular Ca²⁺ regulated NO· formation. Zinc inhibited capacitation and formation of O₂·^? and NO·. Zinc chelators (TPEN and EDTA) and sulfhydryl-targeted compounds (diamide and N-ethylmaleimide) stimulated capacitation and formation of O₂·^? and NO·; superoxide dismutase (SOD) and nitric oxide synthase inhibitor (L-NMMA) prevented these events. Diphenyliodonium (flavoenzyme inhibitor) blocked capacitation and related O₂·^? synthesis but promoted NO· formation, an effect canceled by SOD and L-NMMA. NADPH induced capacitation and NO· (but not O₂·^?) synthesis and these events were blocked by L-NMMA and not by SOD. Integration of these data on O₂·^? and NO· production during capacitation reinforces the concept that a complex, but flexible, network of factors is involved and probably is associated with rescue mechanisms, so that spermatozoa can achieve successful fertilization.     

Decreased superoxide anion radical production by rat alveolar macrophages following inhalation of ozone or nitrogen dioxide

$\text{In vivo}$ exposure of rats to ozone or nitrogen dioxide results in a dose-dependent decrease in superoxide anion radical production (O₂^?·) by alveolar macrophages isolated from the exposed animals. When alveolar macrophages from ozone-exposed animals were stimulated with phorbol myristate acetate (PMA, a non-phagocytic stimulus of O₂^?· production) the decrease in O₂^?· production ranged from 85.9% of control at 3.2 ppm-hrs ozone to 7% of control at 10.5 ppm-hrs. In a similar fashion, O₂^?· production by PMA-stimulated macrophages from NO₂-exposed rates ranged from 78% of control at 18.3 ppm-hrs NO₂ down to 14.5% of control at 51 ppm-hrs. Since the viability of the alveolar macrophages obtained from ozone or nitrogen dioxide-exposed animals was 88% or better in all cases as judged by both Trypan blue exclusion and lactate dehydrogenase release, the decreased ability of these cells to produce superoxide anion radical cannot be attributed to a pollutant effect on cell viability. This diminution in superoxide anion radical production by alveolar macrophages from the pollutant-exposed animals might account, in part, for the ability of these 2 air pollutants to potentiate bacterial infections in laboratory animals.     

Lipid peroxidation and the generation of free radicals, superoxide anion, and hydrogen peroxide in beta-lapachone-treated Trypanosoma cruzi epimastigotes.

β-Lapachone, an antimicrobial agent, was reduced by Trypanosoma cruzi epimastigotes to a semiquinone radical. It markedly increased the generation of superoxide anion and hydrogen peroxide in intact cells. Using NADH as electron donor, β-lapachone also increased significantly the rate of H₂O₂ generation in epimastigote homogenates. Incubation of epimastigotes with β-lapachone stimulated lipid peroxidation.     

Biphasic regulation of superoxide radical levels in Mn-depleted and photoactivated photosystem II

In the past decades, it has become clear that superoxide radical (O₂ ^·?) can be generated from photosystem II (PSII) during photosynthesis. Depending on the extent of its accumulation, O₂ ^·? plays an important role in plant physiology and pathology. The photoinhibition/repair cycle is a typical process in PSII which is mainly responsible for the survival of plants under the photoinihibition condition. It is therefore of significant importance to determine O₂ ^·? production in this cycle, and then explore how O₂ ^·? is controlled by PSII within a normal physiological level. With this in mind, we herein investigate the variation of the O₂ ^·? levels in PSII under Mn-depleted and photoactivated conditions mimicking the photoinhibition/repair cycle in vitro. The effect of intrinsic SOD-like component on the O₂ ^·? levels was also studied. Results show that PSII has the ability to regulate the O₂ ^·? levels in these two processes by simultaneously modulating the O₂ ^·? generation activity and intrinsic SOD-like activity. This finding could shed new lights on the photoprotective property of PSII against O₂ ^·? and other reactive oxygen species.     

The Tiron free radical as a sensitive indicator of chloroplastic photoautoxidation

Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 5–6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and l-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, but added superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.     

Heterologous expression of a halophilic archaeon manganese superoxide dismutase enhances salt tolerance in transgenic rice

In order to investigate new gene resource for enhancing rice tolerance to salt stress, manganese superoxide dismutase gene from halophilic archaeon (Natrinema altunense sp.) (NaMnSOD) was isolated and introduced into Oryza sativa L. cv. Nipponbare by Agrobacterium-mediated transformation. The transformants (L1 and L2) showed some NaMnSOD expression and increased total SOD and CAT activity, which contributed to higher efficiency of ROS elimination under salt stress. The levels of superoxide anion radicals (O ₂ ^·? ) and hydrogen peroxide (H₂O₂) were significantly decreased. In addition, they exhibited higher levels of photosynthesis, whereas lower relative ion leakage and MDA content compared to wild-type plants. Therefore, transgenic seedlings were phenotypically healthier, and heterologous expression of NaMnSOD could improve rice salt tolerance.     

Responses of the antioxidant defense system to drought stress in the leaves of Fargesia denudata seedlings,the staple food of the giant panda

The responses of the antioxidant defense system in plant species to drought stress are still relatively unknown. In order to further understand how the system responds to drought stress, the leaves of Fargesia denudata seedlings were investigated. Antioxidant enzyme activities, antioxidant contents, hydrogen peroxide (H₂O₂), superoxide anion (O ₂ ^·? ) and MDA contents in the seedling leaves were measured under well-watered (WW), moderate drought-stressed (MD), and severe drought-stressed (SD) treatments. Although drought stress significantly increased H₂O₂ and O ₂ ^·? levels in F. denudata leaves, only weak lipid peroxidation was observed. This is attributed to the higher superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) activities in F. denudata leaves during the entire drought period. Reduced and oxidized ascorbate (AsA and DHA) contents were almost not affected by drought except that DHA under SD showed an obvious increase on day 30. Furthermore, reduced glutathione (GSH) content under drought stress significantly decreased, while oxidized glutathione (GSSG) markedly increased under SD on days 30 and 45 as well as under MD on day 30; as a result, the ratio GSH/GSSG declined considerably. These results indicated that GSH was involved in scavenging H₂O₂ and O ₂ ^·? under drought stress and it was more sensitive to drought stress in scavenging H₂O₂ and O ₂ ^·? than AsA. As a result, a highly efficient antioxidant defense system in drought-stressed F. denudate leaves operated mainly through the synergistic functioning of SOD, CAT, APX, MDHAR, DHAR, GR, and GSH against oxidative damage.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Dicarboxylate transport in maize mesophyll chloroplasts

Evidence is presented for high rates of carrier-mediated dicarboxylate anion transport in maize mesophyll chloroplasts. Radioactively labeled malate is transported across the chloroplast envelope leading to accumulation in the stroma. Malate in the stroma will exchange for external malate, oxaloacetate, glutamate, aspartate, and oxoglutarate. At 4 °C the V of malate uptake is 50 μmol·h^?1·mg Chl^?1 and the K_m for malate is 0.5 mm. Oxaloacetate competitively inhibits malate uptake with a K_i estimated to be 0.3 mm. The temperature dependence of malate uptake indicates an activation energy of 12 kcal/mol, and extrapolation using this value gives a rate of transport at 30 °C of approximately 300 μmol·h^?1·mg Chl^?1. This rate approximates the rates of photosynthetic malate production by these chloroplasts.