比色法测定Fenton反应产生的羟自由基

Fenton反应产生的羟自由基能与水杨酸生成羟基化产物2,3－二羟基苯甲酸,用比色法测定其含量能间接测定羟自由基的生成量．通过对测定条件的研究,得到最佳的测定方案．可作为一种简便的筛选羟自由基清除剂的方法     

The effect of pyrophosphate, tripolyphosphate and ATP on the rate of the Fenton reaction

It has been recently reported that pyrophosphate, tri-polyphosphate, ATP and analogous ligands considerably decrease the yield of hydroxyl radicals by the Fenton reaction under conditions where [H₂O₂] > > [Fe(II)L_n]. It was suggested that this effect is due to the slowing down of the Fenton reaction by these ligands. This suggestion seemed surprising as polyphosphate ligands stabilize Fe(III). Indeed, a kinetic study points out that these ligands accelerate the rate of the Fenton reaction by several orders of magnitude. Thus it is suggested that the effect of the ligands on the yield of the hydroxyl radicals is due to the stabilization of the Fe(III) complexes which slows down, or inhibits, their reduction by the radicals formed in the system and thus decreases the overall yield of hydroxyl radicals.     

Formation of Hydroxyl Radicals on Reaction of Hypochlorous Acid with Ferrocyanide, a Model IRON(II) Complex

Hypochlorous acid reacts with the model iron(II) complex, ferrocyanide (Fe(CN)₆^4-) in aqueous solution with the rate constant 220 ± 15 dm³ mol^-1 s^-1. Free hydroxyl radicals are formed in this reaction in 27% yield as shown by the hydroxylation of benzoate to give a product distribution identical to that of free (radiolytically generated) hydroxyl radicals. This reaction is three orders of magnitude faster than the analogous reaction involving hydrogen peroxide (the Fenton reaction), suggesting that the hypochlorous acid generated by activated neutrophils may be a source of hydroxyl radicals.     

Radical graft functional modification of cellulose with allyl monomers: Chemistry and structure characterization

Cotton cellulose was successfully functionalized via a free radical graft polymerization process. Potassium persulfate served as an effective water soluble radical initiator to generate cellulosic radicals. The polymeric radicals could react with allyl monomers such as allyl-dimethylhydantion (ADMH) to form surface grafted cellulose. The reaction sites generated by potassium persulfate were probably at carbon 3 and 4 in glucose ring via oxidative hydrogen abstraction. The cellulosic radicals can initiate grafting polymerization of ADMH with a maximum polymerization degree of about 12 based on LC–MS results. The radical graft polymerization mechanisms were proposed based on LC–ESI/MS analysis. The ideal covalent bonding between cellulose and poly (allyl-dimethylhydantion) (PADMH) ensured permanent graft of the monomers on cotton and durability of the expected functions on the treated cotton.     

Lipid peroxidation initiated by superoxide-dependent hydroxyl radicals using complexed iron and hydrogen peroxide

Iron salts stimulate lipid peroxidation by decomposing lipid peroxides to produce alkoxyl and peroxyl radicals which initiate further oxidation. In aqueous solution ferrous salts produce OH. radicals, a reactive species able to abstract hydrogen atoms from unsaturated fatty acids, and so can initiate lipid peroxidation. When iron salts are added to lipids, containing variable amounts of lipid peroxide, the former reaction is favoured and OH. radicals contribute little to the observed rate of peroxidation. When iron is complexed with EDTA, however, lipid peroxide decomposition is prevented, but the complex reacts with hydrogen peroxide to form OH. radicals which are seen to initiate lipid peroxidation. Superoxide radicals appear to play an important part in reducing the iron complex.     

比色法测定Fenton反应产生的羟自由基及其应用

Fenton反应产生的羟自由基与二甲亚砜反应,生成甲基亚磺酸,再与坚牢蓝BB盐反应生成偶氮砜,比色法测定其含量可间接测定OH·的生成量. 通过对测定条件的研究,得到最佳实验方案. 抗氧化剂药物硫脲和抗坏血酸与羟自由基清除率具有明显的量效关系. 测定了核桃、黑芝麻等几种天然食物的水提取物清除羟自由基的功能. 此法可用于羟自由基清除剂的筛选.     

Do ecto- and ericoid mycorrhizal fungi produce peroxidase activity?

 Several reports attest to the apparent ability of some ectomycorrhizal (ECM) and ericoid (ERM) mycorrhizal fungi to produce peroxidase enzyme activities during growth in axenic culture. In critically reviewing these data, we highlight that apparent peroxidase activities have been observed during growth in media containing 60–70 μM Fe. ECM and ERM fungi are known to produce H₂O₂ via carbohydrate oxidase activity and conditions in common culture media are favourable to the production of hydroxyl radicals, superoxide radicals and ferryl ions via the Fenton reaction. Free radicals so produced can mediate oxidation of substrates commonly used in presumptive peroxidase assays, leading to false-positive results. We argue that there is currently no evidence to support production of peroxidase activity by ECM or ERM fungi, but highlight circumstances in which peroxidase expression might be observed in future work. Accepted: 16 July 1998     

Structure-activity relationship studies of protective function of nitroxides in Fenton system

The aim of this study was to evaluate the effect of piperidine nitroxides and their amine precursors on deoxyribose oxidation in the Fenton system. Protecting activity of nitroxides was found to be concentration-dependent and strongly influenced by ring substituents, while secondary amines did not provide any protection. The reported results suggest a mechanism of nitroxide action through iron oxidation rather than through direct scavenging of hydroxyl radicals. Moreover, presented data point to the danger of interference of nitroxides during the TBARS assay procedure.     

Cysteamine Oxygenase: Possible Involvement of Superoxide Ion in the Catalytic Mechanism

The reaction catalyzed by cysteamine oxygenase on cysteamine in the presence of phenazine methosulphate as cofactor like compound is inhibited by nitroblue tetrazolium, a scavenger of superoxide ions. The reaction is not inhibited by superoxide dismutase and allyl alcohol and it is not activated by superoxide ions produced in solution. Nitroblue tetrazolium is reduced by cysteamine or mercaptoethanol and phenazine methosulphate. This reaction is completely inhibited by superoxide dismutase. In the presence of cysteamine oxygenase the reduction with mercaptoethanol is greatly enhanced and it is only partially inhibited by superoxide dismutase. According to these data a reaction mechanism is proposed in which superoxide ions and thiyl radicals are produced at the active site during catalysis.     

Pathways for production of Fenton's reagent by wood-rotting fungi

Abstract: Many forms of Fe(II) react with H₂0₂ to generate hydroxyl radicals (Fenton reaction). There is evidence that hydroxyl radicals are important in brown-rot, while they can be formed by secondary reactions during lignin breakdown by white-rot fungi. Their involvement in cellulose breakdown creates a range of oxidized sugars. The two reactants of Fenton's reagent can be generated by Fe(II) autoxidation, or by superoxide in reaction with Fe(III). A rapid autoxidation is not possible for complexes with a high Fe(III)/Fe(II) redox potential. Turning to specific pathways for formation of Fenton's reagent, decomposition of Fe(III)-oxalate is probably solely a photochemical process. Lignin peroxidases can act indirectly as a source of superoxide, either by reactions that lead to a peroxyradical, or by 1-electron oxidation of an aliphatic compound creating a strong reductant. Cellobiose dehydrogenase can provide a direct enzymic source for Fenton's reagent (S.M. Kremer and P.M. Wood (1992) Eur. J. Biochem. 208, 807–814). In the experiments as published, hydroxyl radical production was limited by the slow interaction of cellobiose dehydrogenase with O₂. This limitation can be removed by the presence of an iron complex with an autoxidizable Fe(lI) state. The successful use of Fenton's reagent by a living organism requires a spatial separation between initiating enzyme(s) and the site of production of hydroxyl radicals. The mobility of the extra electron on Fe(II) by intermolecular transfer may be important for achieving this separation.     

Copper-Ligand Interactions and Physiological Free Radical Processes. pH-Dependent Influence of Cu2+ Ions on Fe2+-Driven OH- Generation

Prior to comparative studies on the reactivity of various copper complexes with respect to OH radicals, the influence of free Cu²⁺ ions on the superoxide-independent generation of OH radicals through Fenton assays and water gamma radiolysis has been tested in the present work.

Cu²⁺ ions have been shown to behave in a distinct manner towards each of these two production systems. As was logically expected from the noninvolvement of copper in OH^- radical production through gamma radioiysis, no influence of Cu²⁺ ions has been observed on the amount of radicals detected in that case. In contrast, Cu²⁺ ions do influence OH^- radical generation through iron-driven Fenton reactions, but differently depending on copper concentration.

When present in high concentrations, Cu²⁺ ions significantly contribute to OH^- radical production, which confirms previous observations on the reactivity of these in the presence of hydrogen peroxide. At lower levels corresponding to copper/iron ratios below unity on the contrary, Cu²⁺ ions behave as inhibitors of the OH^- production in a pH-dependent manner over the 1-6 range investigated: the lower the pH, the greater the inhibition.

The possible origin of this previously unreported inhibitory effect is discussed.     

Photooxidative reactions in chloroplast thylakoids. Evidence for a Fenton-type reaction promoted by superoxide or ascorbate

A methyl viologen (MV)^* mediated Mehler reaction was studied using Type C and D chloroplasts (thylakoids) from spinach. The extent of photooxidative reactions were measured as (a) rate of ethylene formation from methional oxidation indicating the production of oxygen radicals, and (b) rate of malondialdehyde (MDA) formation as a measure of lipid peroxidation. Without added ascorbate, 1 M FerricEDTA increased ethylene formation by greater than 4-fold, but had no effect on MDA production. Ascorbate (1 mM) produced a tripling of ethylene while it reduced MDA formation in the presence of iron. Radical scavengers diethyldithiocarbamate (DDTC), formate, 1,4-diazabicyclo (2.2.2octane) (DABCO), inhibited ethylene formation. Using 0,4 M mannitol to scavenge hydroxyl radicals, the rates of ethylene formation were reduced 40 to 60% with or without 1 M Fe(III) EDTA. The strong oxidant(s) not scavenged by mannitol are hypothesized to be either alkoxyl radicals from lipid peroxidation, or site specific formation of hydroxyl radicals in a lipophillic environment not exposed to mannitol. Singlet oxygen does not appear to be a significant factor in this system. Catalase strongly inhibited both ethylene and MDA synthesis under all conditions; 1 mM ascorbate did not reverse this inhibition. However, the strong superoxide dismutase (SOD) inhibition of ethylene and MDA formation was completely reversed by 1 mM ascorbate. This suggests that superoxide was functioning as an iron reducing agent and that in its absence, ascorbate was similarly promoting oxidations. Therefore, these oxidative processes were dependent on the presence of H₂O₂ and a reducing agent, suggesting the involvement of a Fenton-type reaction.Abbreviation DABCO 1,4-diazabicyclo(2.2.2.octane) - DCMU 3-(3,4 Dichlorophenyl). 1,1-dimethyl urea - DDTC diethyldithiocarbamate - EDTA ethylenediamine-tetraacetic acid - MDA malondialdehyde - MV methyl viologen - SOD superoxide dismutase - TBA thiobarbituric acid - TCA trichloroacetic acid Scientific contribution number 1315 from the New Hampshire Agriculture Experiment Station.     

Fenton-type reactions and iron concentrations in the midgut fluids of tree-feeding caterpillars

Peroxides are formed in the midgut fluids of caterpillars when ingested tannins and other phenolic compounds oxidize. If these peroxides broke down in the presence of redox-active metal ions, they would form damaging free radicals (Fenton-type reactions). Elemental iron is present in relatively large amounts in leaves and artificial diets, but little is known about its concentration and redox state in midgut fluids, or the extent of Fenton-type reactions in these conditions. This study compared the levels of hydroxyl radicals and iron in the midgut fluids of two species of caterpillars: Orgyia leucostigma, in which phenol oxidation is limited, and Malacosoma disstria, in which phenol oxidation is more extensive. We tested two hypotheses: (1) higher levels of hydroxyl radicals are formed in M. disstria (consistent with the higher concentrations of hydrogen peroxide in this species), and (2) lower concentrations of iron are present in O. leucostigma (providing greater protection of its midgut fluids from oxidative damage). Hydroxyl radical levels increased greatly in M. disstria, but not in O. leucostigma, when they consumed a tannin-containing diet, supporting the first hypothesis. Protein oxidation was also significantly increased in the midgut fluids of M. disstria that ingested tannic acid, consistent with hydroxyl radical damage. Contrary to the second hypothesis, similar concentrations of iron (70 microM) remained in solution or suspension in both species of caterpillars on an artificial diet. Over 90% of this iron appeared to be in the reduced (catalytically active) state in both species. We conclude that tree-feeding caterpillars protect their midgut fluids from oxidative damage caused by Fenton-type reactions by limiting the formation of peroxides, rather than by limiting the availability of reduced iron.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

基于与木糖进行美拉德反应的低聚壳聚糖衍生物的抗氧化性能研究

研究低聚壳聚糖与木糖的美拉德反应,考察了两种体系(低聚壳聚糖与木糖的质量比分别为1∶1和1∶3)反应过程中pH、吸光度及荧光值的变化,醇沉法提取4 h和8 h的低聚壳聚糖美拉德反应衍生物,分别为CX11-4、CX13-4、CX11-8和CX13-8。对衍生物进行红外表征和分子量测定,并研究其对羟基自由基.OH和DPPH的清除能力以及还原能力。结果显示:壳聚糖衍生物的抗氧化能力都明显优于低聚壳聚糖,抗氧化活性顺序为CX13-4>CX11-4,CX11-8>CX13-8。可见,壳聚糖美拉德衍生物的抗氧化活性不仅与反应物的比例有关,还与反应的时间有关。     

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 ^?·.     

A method to evaluate capacity and efficiency of water soluble antioxidants as peroxyl radical scavengers

In this paper, we report on a method to evaluate the activity of water soluble and H-atom donor antioxidants as peroxyl radical scavengers in a micelle system reproducing the conditions occurring in the upper small intestine in humans, during digestion and absorption of lipids. This method, which overcomes some of the problems of the total radical trapping antioxidant parameter (TRAP) assays, measures the peroxyl radical trapping capacity (n) and the peroxyl radical trapping efficiency IC50(-1) of antioxidants, that is the number "n" of peroxyl radicals trapped by one molecule of the studied antioxidant and the reciprocal of the antioxidant concentration that halves the steady-state concentration of peroxyl radicals, respectively. These two fundamental parameters characterizing the radical chain breaking of many water soluble antioxidants, among which dietary polyphenols, can be obtained with relatively good precision from a single experiment, on the basis of a rigorous treatment of the kinetic data.     

Computer simulation of sustained oscillations in peroxidase-oxidase reaction

A system of differential equations of second order exhibiting transitional behaviour and sustained oscillations has been obtained for a complete scheme of the peroxidase-oxidase reaction. The concentrations of hydrogen peroxide and of hydrogen donor radicals are slow variables of the system. The most essential reactions responsible for oscillations have been selected. Analysis of the system in phase plane and in parameter space has been carried out. The dependence of oscillation period and amplitude on the parameter values has been investigated.     

Inhibition of Fe2+- and Fe3+- induced hydroxyl radical production by the iron-chelating drug deferiprone

Deferiprone (L1) is an effective iron-chelating drug that is widely used for the treatment of iron-overload diseases. It is known that in aqueous solutions Fe²⁺ and Fe³⁺ ions can produce hydroxyl radicals via Fenton and photo-Fenton reactions. Although previous studies with Fe²⁺ have reported ferroxidase activity by L1 followed by the formation of Fe³⁺ chelate complexes and potential inhibition of Fenton reaction, no detailed data are available on the molecular antioxidant mechanisms involved. Similarly, in vitro studies have also shown that L1–Fe³⁺ complexes exhibit intense absorption bands up to 800 nm and might be potential sources of phototoxicity. In this study we have applied an EPR spin trapping technique to answer two questions: (1) does L1 inhibit the Fenton reaction catalyzed by Fe²⁺ and Fe³⁺ ions and (2) does UV–Vis irradiation of the L1–Fe³⁺ complex result in the formation of reactive oxygen species. PBN and TMIO spin traps were used for detection of oxygen free radicals, and TEMP was used to trap singlet oxygen if it was formed via energy transfer from L1 in the triplet excited state. It was demonstrated that irradiation of Fe³⁺ aqua complexes by UV and visible light in the presence of spin traps results in the appearance of an EPR signal of the OH spin adduct (TMIO–OH, a(N)=14.15 G, a(H)=16.25 G; PBN–OH, a(N)=16.0 G, a(H)=2.7 G). The presence of L1 completely inhibited the OH radical production. The mechanism of OH spin adduct formation was confirmed by the detection of methyl radicals in the presence of dimethyl sulfoxide. No formation of singlet oxygen was detected under irradiation of L1 or its iron complexes. Furthermore, the interaction of L1 with Fe²⁺ ions completely inhibited hydroxyl radical production in the presence of hydrogen peroxide. These findings confirm an antioxidant targeting potential of L1 in diseases related to oxidative damage.     

Reactive oxygen species regulate oxygen-sensitive potassium flux in rainbow trout erythrocytes

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K(+)-Cl(-) cotransport). 5 mM hydrogen peroxide caused an increase in K(+)-Cl(-) cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K(+)-Cl(-) cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K(+)-Cl(-) cotransport significantly at hypoxic conditions (1% O(2)). These data suggest that hydroxyl radicals, formed from O(2) in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K(+)-Cl(-) cotransporter.