Evidence for Involvement of the Superoxide Radical in the Conversion of 1-Aminocyclopropane-1-Carboxylic Acid to Ethylene by Pea Microsomal Membranes

Electron spin resonance (ESR) spectroscopy has provided evidencefor involvement of the superoxide anion (O₂^–) radicalin the conversion of l-aminocyclopropane-l carboxylic acid (ACC)to ethylene by microsomal membranes from etiolated pea seedlings.Formation of ethylene from ACC by the membrane system is oxygen-dependent,heat denaturable, inhibited by the radical scavenger n-propylgallate and sensitive to superoxide dismutase (SOD) and catalase.Addition of 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron)to the reaction mixture results in formation of the Tiron semiquinone(Tiron radical) ESR signal derived from O₂^–, and alsoinhibits ethylene production. The radical signal is oxygen-dependentand inhibited by SOD and catalase, but is formed both in thepresence and absence of ACC. Heat denaturation of the microsomalenzyme system completely blocks formation of the radical signal.The data collectively suggest that O₂^– generated by amembrane-bound enzyme facilitates the conversion of ACC to ethylene. (Received September 8, 1981; Accepted January 19, 1982)     

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5x10(-7) M) stimulated PMN (6x10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8x10(-6) M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2x10(-5) and 2x10(-4) M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5x10(-6) M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8x10(-6) M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2x10(-6) to 10(-5) M), but N-hexanoyl SPN was less active (from 2x10(-5) to 2x10(-4) M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.     

Formation of protein tyrosine ortho-semiquinone radical and nitrotyrosine from cytochrome c-derived tyrosyl radical

Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.     

Bicarbonate enhances the peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical.

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro-gamma-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H(2)O(2)-mediated nitration of gamma-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H(2)O(2)-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H(2)O(2)-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H(2)O(2), suggest that SOD1/H(2)O(2) oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H(2)O(2) by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226-1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H(2)O(2). However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1).The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.     

Production of singlet oxygen-derived hydroxyl radical adducts during merocyanine-540-mediated photosensitization: analysis by ESR-spin trapping and HPLC with electrochemical detection.

Activated oxygen species produced during merocyanine 540 (MC540)-mediated photosensitization have been examined by electron spin resonance (ESR) spin trapping and by trapping reactive intermediates with salicylic acid using HPLC with electrochemical detection (HPLC-EC) for product analysis. Visible light irradiation of MC540 associated with dilauroylphosphatidylcholine liposomes in the presence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO/.OH). Addition of ethanol or methanol produced additional hyperfine splittings due to the respective hydroxyalkyl radical adducts, indicating the presence of free.OH.DMPO/.OH formation was not significantly inhibited by Desferal, catalase, or superoxide dismutase (SOD). Production of DMPO/.OH was strongly inhibited by azide and enhanced in samples prepared with deuterated phosphate buffer (PB-D2O), suggesting that singlet molecular oxygen (1O2) was an important intermediate. When MC540-treated liposomes were irradiated in the presence of salicylic acid (SA), HPLC-EC analysis indicated almost exclusive formation of 2,5-dihydroxybenzoic acid (2,5-DHBA), with production of very little 2,3-DHBA, in contrast to .OH generated by uv photolysis of H2O2, which gave nearly equimolar amounts of the two products. 2,5-DHBA production was enhanced in PB-D2O and inhibited by azide, again consistent with 1O2 intermediacy. 2,5-DHBA formation was significantly reduced in samples saturated with N2 or argon, and such samples showed no D2O enhancement. Ethanol had no effect on 2,5-DHBA production, even when present in large excess. Catalase and SOD also had no effect, and only a small inhibition was observed with Desferal. DMPO inhibited 2,5-DHBA production in a concentration-dependent fashion and enhanced formation of 2,3-DHBA. We propose that 1O2 reacts with DMPO to give an intermediate which decays to form DMPO/.OH and free.OH, and that the reaction between 1O2 and SA preferentially forms the 2,5-DHBA isomer. This latter process may provide the basis for a sensitive analytical method to detect 1O2 intermediacy. Singlet oxygen appears to be the principle activated oxygen species produced during MC540-mediated photosensitization.     

Superoxide anion and hydroxyl radical scavenging activities of vegetable extracts measured using electron spin resonance.

Radical scavenging by reconstituted lyophilized powders of water extracts from 16 common vegetables was measured using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), hydroxyl radicals, (.OH) or superoxide anion radicals (O2.-), as DMPO-OH or DMPO-OOH spin adducts. On a dry weight basis, eggplant, and red, yellow and green bell pepper extracts showed potent superoxide anion radical scavenging activities (SOD-like activities). Ascorbate oxidase- or heat-treatments, decreased SOD-like activities in bell pepper extracts suggesting that ascorbate accounts for much of their free radical scavenging activity. Eggplant epidermis extract exhibited the most potent hydroxyl radical scavenging and SOD-like activities. Eggplant SOD-like activity did not decrease after ascorbate oxidase treatment, but decreased following ultrafiltration demonstrating that SOD-like activity is partially due to high molecular weight substances. Nasunin, an anthocyanin in eggplant epidermis, showed markedly potent superoxide anion radical scavenging activity, while it inhibited hydroxyl radical generation probably by chelating ferrous ion.     

Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical

The effect of bicarbonate anion (HCO(3)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(3)(-) enhanced SOD/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(3)(-) enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(3)(-)-mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis.     

Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO

The spin trapping ESR technique was applied to investigate oxygen-derived radicals in ischemic and post-ischemic rat hearts. Using 5,5'-dimethyl-l-pyrroline-N-oxide, carbon-centered radicals were identified during ischemia and oxy-radical adducts (superoxide anion radical, O.-2 and hydroxyl radicals, .OH) in post-ischemic rat heart. The formation of these spin adducts was inhibited by superoxide dismutase, suggesting that superoxide plays a role in the adducts' formation. The results demonstrate that oxygen derived free radicals are important byproducts of abnormal oxidative metabolism during myocardial ischemic and reperfusion injuries.     

Kinetic study on ESR signal decay of nitroxyl radicals,potent redox probes for in vivo ESR spectroscopy,caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.     

铜锌超氧物歧化酶对几种羟自由基产生系统的影响

应用脱氧核糖降解法研究了ＣｕＺｎ－ＳＯＤ对几种·ＯＨ产生系统的作用机理．结果证明：ＳＯＤ对Ｆｅ（３＋）·Ｏ·Ｈ２Ｏ２系统中·ＯＨ的产生有明显的抑制作用,而失活ＳＯＤ或ＢＳＡ对它的抑制作用不大;在Ｆｅ（２＋）·Ｈ２Ｏ２和ＣＵ（２＋）·Ｈ２Ｏ２系统中,ＳＯＤ、失活ＳＯＤ和ＢＡＳ均能抑制·ＯＨ的产生;在Ｆｅ（２＋）·Ｏ系统中,ＳＯＤ对·ＯＨ产生作用不大,而失活ＳＯＤ或ＢＳＡ对它有明显的抑制作用．由此推测ＳＯＤ对·ＯＨ形成可能有三方面的影响：１．对Ｏ的清除作用,阻断Ｈａｂｅｒ－Ｗｅｉｓｓ反应;２．对金属离子的络合作用,降低·ＯＨ的产额;３．促进Ｈ２Ｏ２的积累,加快Ｆｅｎｔｏｎ反应．     

Detection of activated O2 species in vitro and in rat lungs by chemiluminescence

This study used chemiluminescence, an "on-line" photon-counting technique, to detect and characterize activated O2 species in vitro and in isolated rat lungs. The sensitivity and specificity of enhanced chemiluminescence for superoxide anion (O2-.) and hydrogen peroxide (H2O2) was evaluated in vitro. The effect of media conditions (such as O2 tension, albumin concentration, and sulfhydryl group availability) on luminescence was assessed in vitro. Xanthine-xanthine oxidase (X-XO) primarily produced superoxide anion in vitro. Enhanced chemiluminescence varied directly with the dose of luminescent probe used and the quantity of activated O2 species administered. The strength of the luminescent signal was also dependent on the concentration of albumin and O2 in the media. Lucigenin was more sensitive than luminol to the presence of O2-. and, unlike luminol, lucigenin did not alter radical production by XO. However, neither luminescent probe was specific for O2-., as both detected H2O2 and O2 in vitro. H2O2-induced chemiluminescence was inhibited by catalase but not superoxide dismutase (SOD), while X-XO-induced luminescence was inhibited by SOD but not catalase. SOD-inhibitable chemiluminescence was a sensitive and specific marker for O2-. production in vitro. Once the sensitivity-specificity of enhanced chemiluminescence was defined in vitro, this technique was used to explore the mechanism by which exogenous X-XO reduced hypoxic vasoconstriction in isolated rat lungs. The vascular paresis, caused by administration of X-XO to the rat lung, resulted from a brief burst of O2-. production rather than a sustained alteration of lung radical levels.     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.     

A comparison of chemical systems for luminometric determination of antioxidant capacity towards individual reactive oxygen species.

The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthinoxidase (HX-XO), hydrogen peroxide-ferrous sulphate (H2O2-FeSO4) and hydrogen peroxide (H2O2) systems by using various concentrations of ROS scavengers, such as superoxide dismutase (SOD), dimethylthiourea (DMTU) or catalase (CAT). Luminol (0.8 mmol/L) was dissolved in a borate buffer, pH 9.0, and was used as a luminophor in the chemiluminescence (CL) measurements. In the HX-XO system SOD, CAT and DMTU deepened the CL signal, whereas in the H2O2-FeSO4 system, only CAT and DMTU deepened the CL signal, and in the H2O2 system SOD and CAT increased and DMTU deepened the CL signal. Electron spin resonance (ESR) measurements were performed only in the H2O2-FeSO4 system. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as a spin trap. According to typical ESR spectra, .OH was produced in this chemical system. It can be concluded that the chemical systems do not produce single reactive oxygen species but a mixture of them.     

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.     

Enhancement by streptozotocin of O2- radical generation by the xanthine oxidase system of pancreatic beta-cells

Spin-trapping techniques and electron spin resonance (ESR) spectroscopy were used to study the relationship between the effect of streptozotocin (STZ) on pancreatic beta-cells and free radical formation by these cells. Results showed that STZ enhanced generation of the DMPO-OH radical adduct, which is a degradation product of the superoxide anion (O2-) in the presence of cellular components, in a hypoxanthine-xanthine oxidase (XOD) system with a homogenate of beta-cells. This enhancing effect was also observed in a system without cellular components; STZ increased the signal height due to the O2- radical in a concentration-dependent manner and caused a maximum of 150% enhancement at a concentration of 1.5 mM. Thus, STZ seemed to enhance the generation of the O2- radical in the XOD system, probably by some mechanism of its interaction with XOD. Pancreatic beta-cells exhibited a high XOD activity and a very low superoxide dismutase activity. Therefore, the present result supports the possibility that the cytotoxic effect of STZ is closely related to free radical generation in pancreatic beta-cells.     

5,5-dimethyl-1-pyrroline-N-oxide alone enhances the spontaneous superoxide generation by primaquine.

Primaquine (PQ), a well-known antimalarial drug, has been reported to generate superoxide (O2-) in the presence of reducing agents such as NADPH. In the present study, chemiluminescence was detected by adding only PQ to aqueous 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-alpha] pyrazin-3-one (MCLA), which is a specific chemiluminescent probe for O2-, and was quenched by superoxide dismutase (SOD), indicating that PQ alone can generate O2- in aerobic conditions. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enhanced the O2- generation by PQ. Superoxide spin adduct, DMPO-OOH, was also detected by ESR both in aqueous solutions and in dimethyl sulfoxide with DMPO. The level of O2- generation showed a linear correlation with the DMPO concentration, and SOD competitively inhibited the DMPO-OOH formation. The results suggested that in aerobic conditions PQ is autoxidized to 5-hydroxy-PQ, which generates O2-, and DMPO accelerates the autoxidation process by trapping O2-. DMPO or M4PO alone enhances the spontaneous O2- generation by PQ, therefore cautious evaluation is necessary in all studies using the ESR/spin trapping technique to elucidate the mechanism of PQ-related radical generation.     

Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.

To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.     

Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture

Extracellularly secreted peroxidases in cell suspension culture of tobacco (Nicotiana tabacum L. cv. Bright Yellow-2, cell line BY-2) catalyse the salicylic acid (SA)-dependent formation of active oxygen species (AOS) which, in turn, triggers an increase in cytosolic Ca2+ concentration. Addition of horseradish peroxidase (HRP) to tobacco cell suspension culture enhanced the SA-induced increase in cytosolic Ca2+ concentration, suggesting that HRP enhanced the production of AOS. The mechanism of peroxidase-catalysed generation of AOS in SA signalling was investigated with chemiluminescence sensitive to AOS and electron spin resonance (ESR) spectroscopy, using the cell suspension culture of tobacco, and HRP as a model system of peroxidase reaction. The results showed that SA induced the peroxidase inhibitor-sensitive production of superoxide and H2O2 in tobacco suspension culture, but no production of hydroxy radicals was detected. Similar results were obtained using HRP. It was also observed that SA suppressed the H2O2-dependent formation of hydroxy radicals in vitro. The results suggest that SA protect the cells from highly reactive hydroxy radicals, while producing the less reactive superoxide and H2O2 through peroxidase-catalysed reaction, as the intermediate signals. The formation of superoxide was followed by that of H2O2, suggesting that superoxide was converted to H2O2. In addition, it was observed that superoxide dismutase-insensitive ESR signal of monodehydroascorbate radical was induced by SA both in the tobacco suspension culture and HRP reaction mixture, suggesting that SA free radicals, highly reactive against ascorbate, were formed by peroxidase-catalysed reactions. The formation of SA free radicals may lead to subsequent monovalent reduction of O2 to superoxide.     

Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.