Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide.

In photosynthetically competent chloroplasts from spinach the quantum requirements for oxygen evolution during CO2 reduction were higher, by a factor often close to 1.5, than for oxygen evolution during reduction of phosphoglycerate. Mass spectrometer experiments performed under rate-limiting light indicated that an oxygen-reducing photoreaction was responsible for the consumption of extra quanta during carbon dioxide assimilation. Uptake of 18O2 during reduction of CO2 was considerably higher than could be accounted for by oxygen consumption during glycolate formation and by the Mehler reaction of broken chloroplasts which were present in the preparations of intact chloroplasts. The oxygen reducing reaction occurring during CO2 assimilation resulted in the formation of H2O2. This was indicated by a large stimulation of CO2 reduction by catalase, but not of phosphoglycerate reduction. Catalase could be replaced as a stimulant of photosynthesis by dithiothreitol or ascorbate, compounds known to react with superoxide radicals. There was no effect of dithiothreitol and ascorbate on phosphoglycerate reduction. A main effect of superoxide radicals and/or H2O2 was shown to be at the level of phosphoglycerate formation. Evidence for electron transport of oxygen was also obtained from 14CO2 experiments. The oxidation of dihydroxyacetonephosphate during a dark period or after addition of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone in the light was studied. The results indicated a link between the chloroplast pyridine nucleotide system and oxygen. Oxygen reduction during photosynthesis under conditions where light is rate limiting is seen as important in supplying the ATP which is needed for CO2 reduction but is not provided during electron transport to NADP. A mechanism is discussed which would permit proper distribution of electrons between CO2 and oxygen during photosynthesis.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Singlet Oxygen-Trapping Reaction as a Method of 1O2 Detection: Role of Some Reducing Agents

The production of singlet oxygen by H₂O₂ disproportionation and via the oxidation of H₂O₂ by NaOCl in a neutral medium was monitored by spin trapping with 2,2,6,6 tetramethyl-4-piperidone (TMPone). The singlet oxygen formed in both reactions oxidized 2,2,6,6 tetramethyl-4-piperidone to give nitroxide radicals. However the production of nitroxide radicals was relatively small considering the concentrations of H₂O₂ and NaOCl used in the reaction systems. Addition of electron donating agents: ascorbate, Fe²⁺ and desferrioxamine leads to an increase in the production of nitroxide radicals. We assumed that a very slow step of the reaction sequence, the homolytic breaking of the O-O bond of N-hydroperoxide (formed as an intermediate product during the reaction of ¹O₂ with TMPone) could be responsible for the relatively small production of nitroxide radicals. Electron donating agents added to the reaction system probably raise the rate of the hydroperoxide decomposition by allowing a more rapid heterolytic cleavage of the O-O bond leading to a greater production of nitroxide radicals. The largest effect was observed in the presence of desferrioxamine. Its participation in this process is proved by the concomitant appearance of desferrioxamine nitroxide radicals. The results obtained demonstrate that the method proposed by several authors and tested in this study to detect singlet oxygen is not convenient for precise quantitative studies. The reactivity of TMPone towards O₂^-7HO₂' and 'OH has been also investigated. It has been found that both O₂^-7HO₂' and 'OH radicals formed in a phosphate buffer solution (pH 7.4, 37°C), respectively by a xanthine-oxidase/hypoxanthine system and via H₂O₂ UV irradiation, do not oxidize 2,2,6,6 tetramethyl-4-piperidone to nitroxide radicals.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.     

Multicomponent Investigations of the Hydrogen Peroxide- and Hydroxyl Radical-Scavenging Antioxidant Capacities of Biofluids: The Roles of Endogenous Pyruvate and Lactate Relevance to Inflammatory Joint Diseases

High field proton (¹H) nuclear magnetic resonance (NMR) analysis of biofluids (healthy human blood sera and inflammatory knee-joint synovial fluids) has been employed to evaluate the hydrogen peroxide (H₂O₂)- and hydroxyl radical (°OH)- scavenging antioxidant capacities of a range of polar, low-molecular-mass endogenous metabolites therein. Data obtained indicate that consumption of H₂O₂ by pyruvate (generating acetate and CO₂ via an oxidative decarboxylation reaction) and °OH radical by lactate (generating pyruvate, and subs quently acetate and CO₂) may serve to protect alternative biofluid components (e.g., macromolecules) against reactive oxygen species-mediated oxidative damage in vivo. The mechanistic, physiological and potential therapeutic implications of these results are discussed with special reference to inflammatory joint diseases.     

CO2加富对UV-B辐射胁迫下亚心形扁藻光合作用和膜脂过氧化以及抗氧化酶活性的影响

在CO2浓度分别为当今CO2浓度(360 μL/L)和加富浓度(5 000 μL/L)条件下,研究了UV-B胁迫对亚心形扁藻(Platymonas subcordiformis(Wille)Hazen)的光合作用、膜脂过氧化和抗氧化酶活性的影响.实验结果表明:(1)UV-B单独作用下,亚心形扁藻的干重、光合速率、叶绿素a(Chl a)和类胡萝卜素(Car.)含量显著降低,CO2加富单独作用下,亚心形扁藻的干重和光合速率显著升高,叶绿素a和类胡萝卜素含量与对照相比没有显著变化,而UV-B与CO2共同作用则使亚心形扁藻的干重和光合速率与对照相比没有显著变化,叶绿素a和类胡萝卜素含量显著降低.(2)UV-B单独作用和CO2加富单独作用都使可溶性蛋白含量显著降低,UV-B与CO2共同作用下的可溶性蛋白含量比UV-B单独作用的要高.高CO2对藻的可溶性蛋白含量的变化在很大程度上归因于Rubisco蛋白的降低.(3)UV-B单独作用下,O-.2产生速率、H2O2含量和MDA含量显著升高,而CO2加富单独作用下,O-.2产生速率、H2O2含量和MDA含量显著降低,与UV-B单独作用相比,UV-B与CO2共同作用使O-.2产生速率、H2O2含量和MDA含量显著降低.说明CO2加富可以减少活性氧对亚心形扁藻的氧化胁迫,同时减少UV-B对亚心形扁藻的膜脂过氧化伤害.(4)UV-B单独作用下,SOD、POD、CAT、GR和GPx活性显著升高,高CO2单独作用使SOD、POD和GR活性显著降低,而CAT和GPx活性与对照相比稍有所降低,但降低不明显,而UV-B与CO2共同作用则使SOD、POD、CAT、GR和GPx活性比UV-B单独作用少得多.结果表明,高CO2对UV-B胁迫所造成的氧化胁迫具有一定的改善作用,因此CO2浓度升高可能对增强海洋微藻的抗逆能力有利.     

Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H₂O₂. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh⁵dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo⁸dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H₂O₂ (200 μM), resulted in a remarkable sensitization of oh⁵dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10⁶ dCyd (15-fold). In contrast, the level of oxo⁸dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10⁶ dGuo (50%). The formation of oh⁵dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H₂O₂. Additional studies showed that replacement of H₂O₂ with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H₂O₂-induced oxidative DNA damage in cells, leading to elevated levels of oh⁵dCyd and oxo⁸dGuo, with a strong bias toward the formation of oh⁵dCyd.     

Inactivation of alcohol dehydrogenase by piroxicam-derived radicals

Alcohol dehydrogenase (ADH) was used as a marker molecule to clarify the mechanism of gastric mucosal damage as a side effect of using piroxicam. Piroxicam inactivated ADH during interaction of ADH with horseradish peroxidase and H₂O₂ (HRP-H₂O₂). The ADH was more easily inactivated under aerobic than anaerobic conditions, indicating participation by oxygen. Superoxide dismutase, but not hydroxyl radical scavengers, inhibited inactivation of ADH, indicating participation by superoxide. Sulfhydryl (SH) groups in ADH were lost during incubation of piroxicam with HRP-H₂O₂. Adding reduced glutathione (GSH) efficiently blocked ADH inactivation. Other SH enzymes, including creatine kinase and glyceraldehyde-3-phosphate dehydrogenase, were also inactivated by piroxicam with HRP-H₂O₂. Thus SH groups in the enzymes seem vulnerable to piroxicam activated by HRP-H₂O₂. Spectral change in piroxicam was caused by HRP-H₂O₂. ESR signals of glutathionyl radicals occurred during incubation of piroxicam with HRP-H₂O₂ in the presence of GSH. Under anaerobic conditions, glutathionyl radical formation increased. Thus piroxicam free radicals interact with GSH to produce glutathionyl radicals. Piroxicam peroxyl radicals or superoxide, or both, seem to inactivate ADH. Superoxide may be produced through interaction of peroxyl radicals with H₂O₂. Thus superoxide dismutase may inhibit inactivation of ADH through reducing piroxicam peroxyl radicals or blocking interaction of SH groups with O₂^-, or both. Other oxicam derivatives, including isoxicam, tenoxicam and meloxicam, induced ADH inactivation in the presence of HRP-H₂O₂.     

ESR Spin Trapping Detection of Hydroxyl Radicals in the Reactions of Cr(V) Complexes with Hydrogen Peroxide

Electron spin resonance (ESR) measurments provide direct evidence for the involvement of Cr(V) in the reduction of Cr(VI) by NAD(P)H. Addition of hydrogen peroxide (H₂O₂) to NAD(P)H-Cr(VI) reaction mixtures suppresses the Cr(V) signal and generates hydroxyl (OH) radicals (as detected via spin trapping), suggesting that Cr(V) reacts with H₂O₂ to generate the OH radicals. Reaction between H₂O₂ and a Cr(V)-glutathione complex. and between H₂O₂ and several Cr(V)-cdrboxylato complexes also produces OH radicals. These results suggest that Cr(V) complexes catalyze the generation of OH radicals from H₂O₂, and that OH radicals might play a significant role in the mechanism of Cr(VI) cytotoxicity.     

Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H₂O₂, and Cu(II)/H₂O₂/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H₂O₂, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.     

The mechanism of the oxidation of ascorbate and Mn by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn²⁺ by isolated chloroplasts was reinvestigated.

2. 2. Our results suggest that ascorbate or Mn²⁺ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn²⁺ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin.

3. 3. In the absence of ascorbate or Mn²⁺ the superoxide formed dismutases either spontaneously or enzymatically producing O₂ and H₂O₂. In the presence of ascorbate or Mn²⁺, however, the superoxide is reduced to H₂O₂ with no formation of O₂. Consequently, in the absence of reducing compounds, in the reaction H₂O to low potential acceptor one O₂ (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn²⁺ or other suitable reductants up to three molecules O₂ can be taken up per four electrons transported.

4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O₂; the addition of ascorbate or Mn²⁺ to this system results in a two fold stimulation of O₂ uptake, with no stimulation of NADPH oxidation. The stimulation of O₂ uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O₂ uptake in the system H₂O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase.

5. 5. In Class II chloroplasts in the system H₂O → NADP upon the addition of ascorbate or Mn²⁺ an apparent inhibition of O₂ evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts.

6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O₂ uptake) should be revised in the upward direction.

亚适宜温光盐环境下油菜素内酯对黄瓜幼苗抗氧化系统及光合作用的影响

研究了24 表油菜素内酯(EBR)对亚适宜温光盐环境下黄瓜幼苗抗氧化系统及光合作用的影响.结果表明: 与对照相比,亚适宜温光盐环境下黄瓜幼苗叶片H₂O₂含量增加,膜脂过氧化程度加剧,膜透性增强,净光合速率(P_n)、气孔导度(g_s)、胞间CO₂浓度(C_i)和蒸腾速率(T_r)分别显著下降39.3%、40.0%、21.2%和47.2%,幼苗干物质积累减少35.9%.外源喷施EBR可提高亚适宜温光盐环境下黄瓜幼苗的抗氧化酶活性,降低H₂O₂含量及膜透性,缓解亚适宜温光盐环境下P_n、g_s、T_r的下降幅度,幼苗干物质积累增加25.9%,生长加快．EBR可通过调节亚适宜温光盐环境下黄瓜幼苗抗氧化性,减少其膜脂过氧化程度,进而维持其较高的光合性能,有效促进了亚适宜温光盐环境下黄瓜幼苗的生长．     

H2S作为下游信号参与SA对低温弱光下黄瓜幼苗光合作用的调控

水杨酸(SA)和硫化氢(H₂S)在调控非生物胁迫下植物生长发育和生理代谢中均起着非常重要的作用,但二者作为信号分子在调控低温弱光下黄瓜光合作用中的互作关系还不清楚。本试验以黄瓜幼苗为试材,分别用SA和硫氢化钠(NaHS,H₂S供体)及其清除剂或抑制剂喷撒叶面,以适宜温光下去离子水处理为对照(CK),研究低温(8 ℃/5 ℃,昼/夜)弱光(100 μmol·m^-2·s^-1)下SA和H₂S对黄瓜幼苗光合作用的调控及互作关系。结果表明: SA可明显增强L-/D-半胱氨酸脱巯基酶(LCD、DCD)活性及其mRNA表达,促进内源H₂S产生;NaHS对苯丙氨酸解氨酶和异分支酸合成酶活性、mRNA表达量及内源SA含量影响不大。SA和NaHS可使低温弱光下黄瓜幼苗的光合速率、气孔导度和蒸腾速率明显提高,胞间CO₂浓度显著降低;同时增强核酮糖-1,5-二磷酸羧化酶、Rubisco活化酶、景天庚酮糖-1,7-二磷酸酯酶和果糖-1,6-二磷酸醛缩酶活性及其mRNA表达,促进光合碳同化;提高光下PSⅡ实际光化学效率和暗下PSⅡ最大光化学效率,从而减轻低温弱光胁迫对黄瓜幼苗的光合机构的损伤和生长量的影响。H₂S清除剂次牛磺酸(HT)可使SA对低温弱光下黄瓜幼苗的光合作用和生长促进效应明显减弱,而SA抑制剂多效唑和氨基茚磷酸对H₂S诱导的黄瓜幼苗光合机构对低温弱光的耐受性无显著影响,说明H₂S作为SA的下游信号,参与调控低温弱光下黄瓜幼苗的光合作用。     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

Trehalose: Protector of antioxidant enzymes or reactive oxygen species scavenger under heat stress?

Trehalose is known to protect membranes and macromolecules. Its accumulation has been implicated in allowing plants to tolerate stress, including heat-shock. However, under heat-shock, it is not clear whether trehalose eliminates reactive oxygen species (ROS) directly or indirectly by protecting antioxidant enzymes. In this study, we initially examined the effects of trehalose on the activities of key antioxidant enzymes, including superoxide dismutases (SODs), ascorbate catalases (CATs), and ascorbate peroxidases (APX) from wheat (Triticum aestivum L.), and then measured the ability of trehalose to scavenge hydrogen peroxide (H₂O₂) and superoxide anions (O₂⁻). Our results indicated that trehalose protected SOD activity slightly. However, it inhibited CAT and APX activities under heat stress, with a little protection of CAT activity (only about 7% promotion) at 22 °C. Moreover, trehalose scavenged H₂O₂ and O₂⁻ greatly in a concentration-dependent manner, reaching the maximal scavenging H₂O₂ rate of 95% and O₂⁻ rate of 78%, respectively, at 50 mM trehalose. These results suggest that trehalose plays a direct role in eliminating H₂O₂ and O₂⁻ in wheat under heat stress.     

A high-activity ATP translocator in mesophyll chloroplasts of Digitaria sanguinalis, a plant having the C-4 dicarboxylic acid pathway of photosynthesis

The effect of exogenous adenine nucleotides on CO₂ fixation and oxygen evolution was studied with mesophyll protoplast extracts of the C₄ plant Digitaria sanguinalis. Exogenous ATP was found to stimulate the rate of pyruvate and pyruvate + oxalacetate induced CO₂ fixation, as well as reverse the inhibition of CO₂ fixation by carbonyl cyanide m-chlorophenyl hydrazone and several electron transport inhibitors. The ATP-dependent stimulation of CO₂ fixation varied from 40 to 70 μmol CO₂ fixed/mg chlorophyll per h, suggesting that ATP was crossing the chloroplast membranes at rates of 80–140 μmol/mg chlorophyll per h, since 2 ATP are required for each CO₂ fixed. Fixation of CO₂ could also be induced in the dark by exogenous ATP, in which case ADP accumulated outside the chloroplasts. This suggests that external ATP is exchanging for internal ADP. In contrast, ADP and AMP were found not to traverse chloroplast membranes, on the basis that neither nucleotide inhibited CO₂ fixation or stimulated oxygen evolution that was limited by available ADP for phosphorylation. Further evidence that ATP can enter the chloroplasts was obtained by direct measurements of the increase in ATP in the chloroplasts due to addition of exogenous ATP in the dark. These studies yielded minimal rates of ATP uptake on the order of 30–40 μmol/mg chlorophyll per h. It is suggested that a membrane translocator exists that specifically transports ATP into the chloroplasts in exchange for ADP. The significance of these findings are considered with respect to the C₄ pathway of photosynthesis.     

Potential of Bioaugmentation and Biostimulation for Enhancing Intrinsic Biodegradation in Oil Hydrocarbon-Contaminated Soil

Studies were conducted using a 10-chamber Micro-Oxymax (Columbus, OH, USA) respirometer to determine the effect of bioaugmentation and biostimulation (by diverse ways of O₂ supply) on enhancing biodegradation of oil hydrocarbons to reduce risk at a former military airport in Kluczewo, Poland. Indigenous or exogenous bacteria bioaugmentation was used to degrade hydrocarbons. Aerated water and/or aqueous solutions of H₂O₂ or KMnO₄ were used to supply O₂. The intrinsic and enhanced biodegradation was evaluated by the O₂ uptake and CO₂ production rates obtained using a linear regression of the cumulative O₂ uptake and CO₂ production curves. Generally, in all cases biodegradation rates enhanced by bioaugmentation were two to four times higher than the rates of intrinsic biodegradation. Moreover, application of indigenous bacteria was more efficient in comparison to the exogenous consortia. The highest CO₂ production rates were achieved when aqueous solution of KMnO₄ was applied, as the increase of CO₂ production rates were about 71% to 97% higher compared to a control. The aqueous solution of H₂O₂ did not cause any significant improvement of the biodegradation rates. Compared to a control, the addition of aerated water resulted in a decrease of CO₂ production rates. Most probably the excessive soil moisture could reduce the air-filled porosity and, consequently, the oxygen contents in soil.     

Redox Cycling of Human Methaemoglobin by H2O2 Yields Persistent Ferryl Iron and Protein Based Radicals

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H₂O₂, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H₂O₂ resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H₂O₂ showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H₂O₂. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalatic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10 fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: ∼20 mol H₂O₂ and abolished the protein radical steady state. The biological implications for these results are discussed.     

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.