Superoxide Dismutase Enhanced the Formation of Hydroxyl Radicals in a Reaction Mixture Containing Xanthone under UVA Irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 μM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O₂, H₂O₂, and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O₂ ^?·.     

Ferric Ion and Oxygen Reduction at the Surface of Protoplasts and Cells of Acer pseudoplatanus

This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O^?₂, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H₂O₂ that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H₂O₂ (catalase) or preventing H₂O₂ formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O^?₂, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H₂O₂ that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction).     

Molecular Characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica juncea</Emphasis> Cu/Zn-Superoxide Dismutases Reveals Their Regulation of Shoot Regeneration

Superoxide dismutases (SODs) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals (O₂^-) to molecular oxygen (O₂) and hydrogen peroxide (H₂O₂). In this study we characterized an Arabidopsis thaliana CuZnSOD (CSD1), a close ortholog of a previously identified Brassica juncea CuZnSOD (MSOD1). CSD1 and other two homologs CSD2 and CSD3 were spatially regulated in Arabidopsis, and CSD1 exhibited distinct expression patterns in response to different stress treatments. To investigate the in vivo function of SOD, transgenic Arabidopsis plants, expressing sense and antisense MSOD1 RNAs, were generated and those with altered SOD activity were selected for further characterization. Although SOD transgenic plants exhibited normal phenotypes, the shoot regeneration response in transgenic explants was significantly affected by the modulated SOD activity and the corresponding H₂O₂ levels. Transgenic explants with downregulated SOD activity were poorly regenerative, whereas those with upregulated SOD activity were highly regenerative. These results suggest that shoot regeneration in vitro is regulated by the SOD activity.     

ANTIOXIDANT ENZYME RESPONSE AND REACTIVE OXYGEN SPECIES PRODUCTION IN MARINE RAPHIDOPHYTES1

Raphidophytes (class Raphidophyceae) produce high levels of reactive oxygen species (ROS), yet little is known regarding cellular scavenging mechanisms needed for protection against these radicals. Enzymatic activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in conjunction with the production of superoxide (O₂^??) and hydrogen peroxide (H₂O₂) in batch cultures of five different raphidophytes species during early exponential, late‐exponential, and stationary growth phases. The greatest concentrations of O₂^?? per cell were detected during exponential growth with reduced levels in stationary phases in raphidophytes Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara, Chattonella marina (Subrahman.) Y. Hara et Chihara, and Chattonella antiqua (Hada) Ono (strain 18). Decreasing trends from exponential to stationary phases for SOD activity and H₂O₂ per cell were observed in all species tested. Significant correlations between O₂^?? per cell and SOD activity per cell over growth phase were only observed in three raphidophytes (Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua strain 18), likely due to different cellular locations of externally released O₂^?? radicals and intracellular SOD enzymes measured in this study. CAT activity was greatest at early exponential phase for several raphidophytes, but correlations between H₂O₂ per cell and CAT activity per cell were only observed for Fibrocapsa japonica Toriumi et Takano, Chattonella antiqua (strain 18), and Chattonella subsalsa Biecheler. Our results suggest that SOD and CAT play important protective roles against ROS during exponential growth of several raphidophytes, while other antioxidant pathways may play a larger role for scavenging ROS during later growth.     

Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation

The oxidation of NADH and accompanying reduction of oxygen to H₂O₂ stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H₂O₂ all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H₂0₂-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

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.     

Esr Spectra of Radicals of Single-Stranded and Double-Stranded DNA in Aqueous Solution. Implications for Oh-Induced Strand Breakage

In situ photolysis at 20^oC (argon plasma light source, $, $ 200 mm) of oxygen-free solutions containing 2mM H₂0₂ and heat-denatured, single-stranded (sS)DNA from calf-thymus resulted in the ESR spectra of the 6-hydroxy-5,6-dihydro-thymin-5-yl {1} and 5-methyleneuracil {3} radicals linked to the sugar-phosphate backbone. They were generated by reaction of OH radicals with DNA. By comparison of the decay characteristics of the ESR signals with rate constants from pulse-conductivity measurements [E. Bothe, G.A. Qureshi and D. Schulte-Frohlinde, Z. Naturforsch. 38c 1030, (1983)] the thymine-derived radicals {1} and {3} can be excluded as precursors of the fast, dominating component of strand breakage of ssDNA. In the absence of H₂0₂ from native, doubie-stranded (ds)DNA an ESR signal was obtained (singlet, g ～ 2.004, $1/2 ～ 0.8 mT) which was assigned to the deprotonated guanine radical cation, {G'(-H)} of a DNA subunit. It is assumed that by the UV irradiation the guanine radical cation, {G⁺}, is generated, either by monophotonic photoionisation or by electron transfer to pyrimidine bases. By rapid transfer of the bridging proton from {G⁺} to the hydrogen bonded cytosine {G'(-H)} is formed. When photolysis of dsDNA was carried out in the presence of H₂0₂, reaction of photolytically generated OH resulted in peroxyl radicals and purine radicals. The oxygen for formation of the peroxyl radicals is probably produced by reaction of {G' (-H)} with H₂0₂. Photolysis of N₂0-saturated solutions containing dsDNA or ssDNA provided another possibility of generation of OH radicals. Under those conditions the OH-induced radicals {1} and {3} were obtained not only from ssDNA but also from dsDNA.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Antioxidant Effects of Rhodoxanthin from Potamogeton crispus L. on H2O2-Induced RAW264.7 Macrophages Cells

Potamogeton crispus L. (P. crispus) is the type of a widely distributed perennial herbs, which is rich in rhodoxanthin. In this research work, five antioxidant indexes in vitro were selected to study the antioxidant activity of rhodoxanthin from P. crispus (RPC). A model of hydrogen peroxide (H₂O₂) -induced oxidative damage in RAW264.7 cells was established to analyze the antioxidant effect and potential mechanism of RPC. The levels of ROS, MDA and the activities of oxidation related enzymes by H₂O₂ were determined by enzyme linked immunosorbent assay (ELISA). The mRNA expression of Nrf-2, HO-1, SOD1 and SOD2 was measured by qRT-PCR assay. According to the results, RPC had free radical scavenging ability for 2, 2-diphenyl-1-trinitrohydrazine (DPPH), 2,2’-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid radical ion) (ABTS), hydroxyl radical and superoxide anion. RPC significantly decreased the level of MDA and ROS and LDH activity, while increased GSH level and activities of SOD, GSH−Px and CAT. It was showed that RPC could increase the mRNA expression of Nrf-2, HO-1, SOD1 and SOD2 in RAW264.7 cells in a dose-dependently manner. In summary, RPC treatment could effectively attenuate the H₂O₂-induced cell damage rate, and the mechanism is related to the reduction of H₂O₂ induced oxidative stress and the activation of Nrf-2 pathway.     

Nitroxide Sod-Mimics: Modes of Action

Low molecular weight superoxide dismutase mimics have been shown to afford protection from oxidative damage. Such SOD-mimics can readily permeate cell membrane achieving sufficiently high levels both inside and outside the cell to effectively detoxify intracellular O^?₂. Preliminary findings also indicated that metal-based and metal-free SOD-mimics can protect hypoxic cells from H₂O₂-induced damage. The present study explored the possibility that SOD-mimics such as desferrioxamine-Mn(III) chelate [DF-Mn] or cyclic nitroxide stable free radicals could protect from O^?₂-independent damage. Killing of monolayered V79 Chinese hamster cells was induced by H₂O₂ or by t-butyl hydroperoxide (t-BHP) and assayed clonogenically. Neither catalase nor native SOD protected the cells from t-BHP. In contrast. both DF-Mn and cyclic nitroxides protected suggesting cytotoxic processes independent of O^?₂ or of O^?₂ -derived active species. The inhibition of the damage by both metal-free and metal-based SOD mimics is attributable to either SOD-mimic reacting with reduced transition metal to block the Fenton reaction and/or intercepting and detoxifying intracellular organic free radicals.     

Oxygen reduction in a plastoquinone pool of isolated pea thylakoids

Oxygen uptake in isolated pea thylakoids in the presence of an inhibitor of plastoquinol oxidation by b ₆/f-complex dinitrophenylether of 2-iodo-4-nitrothymol (DNP-INT) was studied. The rate of oxygen uptake in the absence of DNP-INT had a distinct maximum at pH 5.0 followed by a decline to pH 6.5 and posterior slow rise, while in the presence of an inhibitor it increased at an increasing pH from 4.5 to 6.5 and then kept close to the rate in its absence up to pH 8.5. Gramicidin D substantially affected the oxygen uptake rate in the absence of DNP-INT, and only slightly in its presence. Such differences pointed to the presence of special oxygen reduction site(s) in photosynthetic electron transport chain `before' cytochrome complex. Oxygen uptake in membrane fragments of Photosystem II (BBY-particles) was low and did not depend on pH. This did not support the participation of Q_B in oxygen reduction in DNP-INT-treated thylakoids. Oxygen uptake in thylakoids in the presence of DNP-INT was inhibited by DCMU as well as by catalase in whole pH range. The catalase effect indicated that oxygen uptake was the result of dioxygen reduction by electrons derived from water, and that H₂O₂ was a final product of this reduction. Photoreduction of Cyt c in the presence of DNP-INT was partly inhibited by superoxide dismutase (SOD), and this pointed to superoxide formation. The latter was confirmed by a rise of the oxygen uptake rate in the presence of ascorbate and by suppression of this rise by SOD. Both tests showed that the detectable superoxide radicals averaged 20–25% of potentially formed superoxide radicals the quantity of which was calculated from the oxygen uptake rate. The obtained data implies that the oxygen reduction takes place in a plastoquinone pool and occurs mainly inside the membrane, where superoxide can be consumed in concomitant reactions. A scheme for oxygen reduction in a plastoquinone pool in thylakoid membranes is proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pistacia lentiscus fruit oil reduces oxidative stress in human skin explants caused by hydrogen peroxide

We investigated the efficacy of Pistacia lentiscus fruit oil (PLFO) for protecting human skin from damage due to oxidative stress. PLFO contains natural antioxidants including polyphenols, sterols and tocopherols. We compared the antioxidant potential of PLFO with extra virgin olive oil (EVOO). Explants of healthy adult human skin were grown in culture with either PLFO or EVOO before adding hydrogen peroxide (H₂O₂). We also used cultured skin explants to investigate the effects of PLFO on lipid oxidation and depletion of endogenous antioxidant defense enzymes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) one day after 2 h exposure to H₂O₂. We found that PLFO scavenged radicals and protected skin against oxidative injury. PLFO exhibited greater antioxidant and free radical scavenging activity than EVOO. Skin explants treated with PLFO inhibited H₂O₂ induced MDA formation by inhibition of lipid oxidation. In addition, the oil inhibited H₂O₂ induced depletion of antioxidant defense enzymes including GPx, SOD and CAT. We found that treatment with PLFO repaired skin damage owing to its antioxidant properties.     

Effects of Free Radicals on the Fluidity of Myocardial Membranes

Free radicals, including superoxide anions (O₂^?_?), hydroxyl radical (HO'), and hypohalite radical (OCl'), as well as oxidants such as hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl), have been indicated in the pathogenesis of myocardial ischemic and reperfusion injury. In this report, we compared the integrity of the myocardial membrane when exposed to these free radicals/oxidants. Isolated rat heart membrane preparations were exposed to chemically generated free radicals with or without their respective scavengers. Membrane fluidity was monitored by fluorescence polarization using the diphenylhexatriene probe, as well as by electron spin resonance (ESR) spectroscopy using 2,2,6,6-tetramethyl piperidine-n-oxyl as the spin labeling agent. HO', H₂O₂, and OCl' + HOCl increased the fluorescence polarization (FP) and microvis-cosity significantly by 1.7-fold, 1.8-fold, and 1.7-fold, respectively, as compared to an only 1.2– fold increase in FP by O₂^?_? O₂^?_? did not alter the fatty acid profiles of the membrane phospholipids. However, HO' and H₂O₂ reduced the arachidonic acid contents in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). These radicals also stimulated the lipid peroxidation by several-fold, while that by O₂^?_? was only insignificant. These results suggest that HO' and H₂O₂ decreased the membrane fluidity and induced lipid peroxidation by releasing the arachidonic acid from PC, PE. and PI.     

Quantification of superoxide radical production in thylakoid membrane using cyclic hydroxylamines

Applicability of two lipophilic cyclic hydroxylamines (CHAs), CM-H and TMT-H, and two hydrophilic CHAs, CAT1-H and DCP-H, for detection of superoxide anion radical (O₂^∙−) produced by the thylakoid photosynthetic electron transfer chain (PETC) of higher plants under illumination has been studied. ESR spectrometry was applied for detection of the nitroxide radical originating due to CHAs oxidation by O₂^∙−. CHAs and corresponding nitroxide radicals were shown to be involved in side reactions with PETC which could cause miscalculation of O₂^∙− production rate. Lipophilic CM-H was oxidized by PETC components, reducing the oxidized donor of Photosystem I, P₇₀₀⁺, while at the same concentration another lipophilic CHA, TMT-H, did not reduce P₇₀₀⁺. The nitroxide radical was able to accept electrons from components of the photosynthetic chain. Electrostatic interaction of stable cation CAT1-H with the membrane surface was suggested. Water-soluble superoxide dismutase (SOD) was added in order to suppress the reaction of CHA with O₂^∙− outside the membrane. SOD almost completely inhibited light-induced accumulation of DCP^∙, nitroxide radical derivative of hydrophilic DCP-H, in contrast to TMT^∙ accumulation. Based on the results showing that change in the thylakoid lumen pH and volume had minor effect on TMT^∙ accumulation, the reaction of TMT-H with O₂^∙− in the lumen was excluded. Addition of TMT-H to thylakoid suspension in the presence of SOD resulted in the increase in light-induced O₂ uptake rate, that argued in favor of TMT-H ability to detect O₂^∙− produced within the membrane core. Thus, hydrophilic DCP-H and lipophilic TMT-H were shown to be usable for detection of O₂^∙− produced outside and within thylakoid membranes.     

In Vztro Scavenger Effect of Dihydroquinoline Type Derivatives in Different Free Radical Generating Systems

The non-toxic and water soluble dihydroquinoline type antioxidants: CH 402 (Na-2,2-dimethyl-l.2-dihydroquinoline-4-yl methane sulphonate) and MTDQ-DA (6.6-methylene bis 2.2-dimethyl-4-methane sulphonicacid: Na-1.2-dihydroquinoline) were studied in various in vitro tests in which oxygen free radicals were generated. Both compounds were shown to scavenge superoxide radical anions O^?₂ produced in aqueous solution by pulse radiolysis with rate constants k (O^?₂ + MTDQ-DA) = 4.10⁸dm³ mol^?1s^?1 and k(O^?₂ +CH402) = 1.5.10⁷dm³ mol^?1s ^?1. CH 402 and MTDQ-DA reduced the H₂O₂ produced in the glucose-glucose oxidase reaction, which was detected by the luminol + hemin reaction with a chemilumi-nometric method. The dihydroquinoline type substrates inhibited the NADPH-induced and Fe^{3 +}—stimulated lipid peroxidation and the ascorbic acid-induced non-enzymatic peroxidation pathways in microsomal fractions of rat and mouse liver.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Oxy Radicals in the Eye Tissues of Rabbits After Diquat In Vivo

It is our hypothesis that oxygen free radicals are the triggering agents in cataractogenesis. However, besides H₂O₂ there is no direct evidence of generation of oxy radicals in the eye tissues. Due to extremely short life of O^?₂, and OH. it is not possible to measure their cellular steady state levels. We found that indirect spectrophotometric techniques based on superoxide dismutase (SOD)-inhibitable cytochrome c reduction for estimation of O^?₂. salicylate hydroxylation for OH. and peroxidase catalysed reoxidation of 2,6-dichlorophenolindophenol for H₂O₂ were suitable, sensitive and reproducible for measurements of the reactive species of O₂ produced in the eye tissues by oxy radical enhancer, diquat in the rabbit eye in vivo, After a single intravitreal injection of 60,120 or 300 nmole diquat in the right eyes, there was a dose-dependent rise in O^?₂ levels, 106–265 fold in the aqueous humor, 34–87 fold in the vitreous humor, 6–19 fold in the lens, and 43–88 fold in the retina as compared to 0.16 μM. 0.21 μM, 2.47 nmole/g and 5.56 nmole/g in tissues of the normal eyes, respectively. There were similar increases of OH * in the eye tissues, and of H₂O₂ in the aqueous humor and vitreous humor after diquat injection.

We propose that endogenous reductants of the eye tissues univalently reduce diquat to its free radical which spontaneously reacts with O₂ generaiing O^?₂, in excessive amounts, further giving rise to H₂O₂ and OH triggering cataractogenesis.     

SUPEROXIDE DISMUTASE ACTIVITY IN PERIDINIUM GATUNENSE IN LAKE KINNERET: EFFECT OF LIGHT REGIME AND CARBON DIOXIDE CONCENTRATION1

The activity of superoxide dismutase (EC 1.15.1.1, superoxide: superoxide oxidoreductase) (SOD) was determined in Peridinium gatunense Lemm. under natural and controlled conditions. SOD activity increased toward the end of the spring algal bloom in Lake Kinneret simultaneously with maximal photosynthetic activity and conditions of elevated ambient stress such as high irradiance. Activity staining of native polyacrylamide gel electrophoresis gels of bloom samples showed a similar pattern to the spectrophotometrically measured SOD. Both Mn SOD and CuZn SOD were present, however no Fe SOD was found in Peridinium. One of three isoenzymes of Mn SOD showed marked differential regulation of activity under stress. An increase in the quantity of the 32-kDa Mn SOD polypeptide during the bloom was found to be unrelated to senescence; it was assumed that this polypeptide was induced by stress. Thus, SOD in Peridinium undergoes physiological and molecular acclimation to seasonal environmental changes. When Peridinium was exposed to various O₂ and CO₂ concentrations in culture, CuZn SOD significantly increased under high C0₂ concentrations and normoxic conditions (20% O₂). However, at high irradiances, Peridinium cultures exposed to low and high CO₂ concentrations also had similar CuZn SOD activity. It was concluded that stressful irradiance is the overriding cause of increased SOD activity in both lake samples and in cultures of Peridinium.     

Steady-state kinetics, micellar effects, and the mechanism of peroxidase-catalyzed oxidation of n -alkylferrocenes by hydrogen peroxide

 Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C₅H₁₁) by H₂O₂ to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25  °C. The rate of oxidation of ferrocenes with longer alkyl radicals is too slow to be measured. The reaction obeying the [RFc]:[H₂O₂] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic second-order rate constants k _w are (1.9 ± 0.5)×10⁵, (2.7 ± 0.1)×10⁴, and (5.9 ± 0.6)×10³ M^–1 s^–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple in terms of the Marcus formalism. Received: 15 July 1996 / Accepted: 15 November 1996