Hydroxyl radical formation via iron-mediated Fenton chemistry is inhibited by methylated catechols

The differing effects of O-methylated catecholamines and their dihydroxyphenyl precursors on the production of ^?OH were quantified using a previously established specific salicylate hydroxylation assay in conjunction with a sensitive electrochemical detection system. The production of ^?OH by the Fenton reaction was diminished significantly by O-methylated catecholamines (O-methyldopa, O-methyldopamine, O-methyltyrosine, and N-acetyl-O-methyldopamine), whereas radical production was augmented by dihydroxyphenyls (DOPA, dopamine, and N-acetyldopamine), including those with methylated side chains (N-methyldopamine and α-methyldopa). Monohydroxyphenyls such as octopamine, tyramine, tyrosine, and α-methyltyrosine had little or no effect on radical production. These data show that a methyl group positioned on the side chain of a catecholamine does not alter its pro-oxidant behavior, while a methyl group positioned on the aromatic ring renders the catecholamine sterically or kinetically unfavorable for coordination with transition metals, thus preventing the promotion of Fenton chemistry. These results highlight the importance of O-methylation in forming catechols that are less reactive than their dihydroxyphenyl precursors. Thus, factors regulating the methylation of brain catecholamines may play a crucial role in mediating neuronal integrity during aging and in the pathogenesis of certain neurodegenerative disorders. Competitive side-chain methylation reactions may sustain or perpetuate some dihydroxyphenyls, creating an oxidatively less favorable environment for cells than would result from compounds formed by O-methylation.     

Chemiluminescence of the Fenton reaction and a dihydroxybenzene-driven Fenton reaction

A dihydroxybenzenes(DHB)-driven Fenton reaction was found to be more efficient than a simple Fenton reaction based on OH radical and activated species production. The reason for this enhanced reactivity by [Fe DHB] complexes is not well understood, but results suggest that it may be explained by the formation of oxidation species different from those formed during the classic Fenton reactions. In previous work, greater concentrations, and more sustained production of OH over time were observed in DHB driven Fenton reactions versus neat Fenton and Fenton-like reactions. In this work, chemiluminescence (CL) was monitored, and compared to OH production kinetics. The CL of the DHB-driven Fenton reaction was shorter than that for sustained production of OH. CL appears to have been caused by excited Fe(IV) species stabilized by the DHB ligands initially formed in the reaction. Formation of this species would have to have occurred by the reaction between OH and Fe(III) in a DHB complex.     

Density Functional Geometry Optimization and Energy Calculations of Calcium(II)-Triphosphate Complexes. Polyphosphates as Possible Dissolving Agents for Calcium Pyrophosphate Dihydrate Crystals in Chondrocalcinosis Disease

Abstract

Geometry optimizations and energy calculations have been carried out via molecular orbital methods at the density functional B3LYP/LANL2DZ level on the molecules PO₃ ^?, OPO₃ ^3-, HOPO₃ ^2-, CH₃OPO₃ ^2-, H(CH₃OPO₃ ^?, O(PO₃)₂ ^4-, HO(PO₃)₂ ^3-, CH₂ (PO₃)₂ ^4-, (CH₃OPO₂)O(PO₃)^3-, O(PO₃)₃ ^5-, HO(PO₃)₃ ^4-, (PO₃)₃ ^3-, (CH₃OPO₂)O(PO₃)₂ ^3-, [Mg{O(PO₃)₂}]^2-, [Ca{O(PO₃)₂}]^2-, [Ca{CH₂ (PO₃)₂}]^2-, [Ca{CH₃OPO₂)O(PO₃)}]^?, [Ca(PO₃)₃]^?, [Ca{O(PO₃)₃}]^3-, and [Ca{CH₃OPO₂)O(PO₃)₂}]^2- with the aim to find reliable and easily accessible computational methods to simulate some phosphate-containing molecules of importance for the living cells and to study the energetics for protonation and metal-complex formation reactions. The analysis is part of a general investigation on phosphate-containing molecules as potential dissolving agents for calcium pyrophosphate dihydrate (CPPD) crystals which deposit in certain articular diseases. The basis set was expanded to 6–31G** for the P atoms for all the molecules investigated and to 6–31G* for the O atoms for OPO₃ ^3-. Calculations at the semi- empirical MNDO/d level were also carried out for comparison purposes on the free ligand molecules and on [Mg{O(PO₃)₂}]^2-. The density functional analysis reproduced well the geometry found at the solid state via X-ray diffraction. The analyses of the geometrical parameters and the total electronic energy of the molecules shows that O(PO₃)₂ ^4- and other di- and tri-phosphates are versatile ligands for divalent metal ions like Ca²⁺. The computed P-O-P bond angle for free O(PO₃)₃ ^4- is 180° and the conformation of the two PO₃ ^? groupings is staggered along the P…P vector. The linear arrangement for P-O-P is assisted by P-Oπ interactions. The bending of the P-O-P angle when accompanied by a slight P-O(b) elongation requires a very small amount of energy; 4.65 kcal/mol to pass from 180 to 140°, as calculated at the DFT level. The computed Ca-O and Mg-O bond distances for [M{O(PO₃)₂}]^2- are 2.378 and 2.079Å, when the metal ions link two oxygen atoms from each PO₃ group. The computed Ca-O bond lengths for [Ca{CH₃OPO₂)O(PO₃)}]^? are 2.482 (P_αO₂) and 2.358Å (P_βO₂), showing a significant lengthening for Ca-OP_α, when compared to the pyrophosphate derivative. The Ca-O bond lengths for [Ca{O(PO₃)₃}]^3- and [Ca{CH₃OPO₂)O(PO₃)2}]^2- are 2.251Å and 2.525 (P_αO₂), 2.407 and 2.338 (P_βO₂), and 2.251 and 2.228Å (P_γO₂), showing a shortening for the Ca-OPγ bond upon methylation. The (P_β)O-P_γ bond length increases significantly (0.09 Å) upon Ca(II) coordination to (CH₃OPO₂)O(PO₃)₂ ^4- via all the three PO₃ groups. This latter result suggests that metal complexes of linear organic-triphosphates have a larger tendency to release the P_γO₃ group when compared to the free ligand molecules. The electronic contribution to the energy of the complex formation reaction for [Ca{CH₂ (PO₃)₂}]^2- is only slightly higher (some 1.8 kcal) than that for [Ca{O(PO₃)₂}]^2-; but is much higher (some 63 kcal) than that relevant to the formation of [Ca{CH₃OPO₂)O(PO₃)₂}]^2-. Therefore the linear methyltriphosphates (and reasonably nucleoside triphosphates) are predicted to form metal complexes which are less stable than diphosphonate or inorganic diphosphate ligands, at least in the case the organic moiety does not have any appreciable coordination ability toward the metal. The energy of complex formation for [Ca{O(PO₃)₃}]^3- is much higher than that for [Ca{O(PO₃)₂}]^2- and [Ca(PO₃)₃}]^? (some 116 and 345 kcal, respectively). This is in part a rationale for the high dissolving ability (DA) toward CPPD crystals, shown by solutions of pentasodium tripolyphosphate. DA values of 65.4±3.0 μg Ca/mL were detected, via atomic absorption spectroscopy, in solutions of Na₅{O(PO₃)₃} 0.06 M after digestion (1.00 h, 37°C, pH 7.4) in the presence of CPPD crystals. Efficient non-toxic dissolving agents for CPPD crystals are needed to cure chondrocal-cinosis desease. The semi-empirical computations gave correct overall optimized structures and conformations for, at least, the free not-substituted ligands and the relevant Mg(II)-com- plexes. The optimized structure for [Mg{O(PO₃)₂}]^2- has computed P-O(t) and P-O(d) bond distances of 1.503 and 1.598Å, in perfect agreement with the results from DFT calculations.     

芽胞抗力快速弱化技术研究

以L-丙氨酸缓冲液为发芽剂,结合芬顿反应原理,观察发芽-氧化损伤效应对芽胞的杀灭效果,以期为新型炭疽疫源地净化方法的深入研究奠定基础。以腊样芽胞为试验菌,采用透射电镜、激光扫描共聚焦显微镜、活菌计数等方法观察芽胞发芽过程的超微结构、核酸含量变化,以及在芬顿反应的联合作用下发芽体的活性变化。在20~30 min的发芽过程中,芽胞核心密度降低,核心与皮质、皮质与外壁之间界限模糊,芽胞外壁和芽胞衣有破裂,通透性增加,进一步有皮质消失、细胞核与细胞质融合、细胞膜基本形成的现象;发芽体荧光强度不断增加,显示菌体中核酸的活性和含量不断增加;发芽体对化学因子的抗力明显下降,H2O2浓度为0.20 mol/L的Fenton反应系统作用60 min时,发芽体灭活可达到3.016个对数级。诱导发芽和反应的联合处理程序可显著提高芽胞的灭活水平。     

Oxidative damage of DNA induced by the reaction of methylglyoxal with lysine in the presence of ferritin

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. In this study, we investigated whether ferritin enhances DNA cleavage by the reaction of MG with lysine. When plasmid DNA was incubated with MG and lysine in the presence of ferritin, DNA strand breakage was increased in a dose-dependent manner. The ferritin/MG/lysine system-mediated DNA cleavage was significantly inhibited by reactive oxygen species (ROS) scavengers. These results indicated that ROS might participate in the ferritin/MG/lysine system-mediated DNA cleavage. Incubation of ferritin with MG and lysine resulted in a time-dependent release of iron ions from the protein molecules. Our data suggest that DNA cleavage caused by the ferritin/MG/lysine system via the generation of ROS by the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin. [BMB Reports 2013; 46(4): 225-229]     

Degradation of Tribromophenol by Wood-Decaying Fungi and the 1,2-Dihydroxybenzene-Assisted Fenton Reaction

The degradation of 2,4,6-tribromophenol (TBP) by biological and chemical treatments was studied. Biological treatment involved the use of Laetoporeus sulfureus, Gloephyllum trabeum, and Ganoderma australe in liquid culture. Despite the inhibitory effects of TBP on the fungal growth, these fungi were able to degrade TBP after 15 days of biotreatment. At 66, 116, and 183 μ M TBP, the degradation by G. australe was the most efficient (71% to 77%), whereas G. trabeum and L. sulfureus degraded between 50% and 60% of three TBP concentrations. The removal of organic bromine reached values of 50% in all cases. The chemical treatment (1,2-dihydroxybenzene-assisted Fenton reaction) achieved up to 90% of TBP degradation. However, only 40% of TBP was mineralized and the toxicity level did not undergo changes during the chemical treatment. On the other hand, a 30% reduction in toxicity was obtained with a combined chemical-biological treatment.     

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.     

Photo-Fenton氧化法处理废水的原理及影响因素

Photo-Fenton高级氧化技术是处理难降解有毒有机废水的一种有效的方法。本文阐述了该氧化法的原理及其影响因素,photo-Fenton氧化法在反应中会产生大量羟自由基(·OH),它是一种非常活泼及非选择性物种,其氧化电位为2.8V,氧化能力很强,能够引发水溶液中大部分有机物的氧化还原反应。其优点是操作简便及无二次污染等,反应产物Fe³⁺可与OH反应形成Fe(OH)₃沉淀而对环境无害。缺点是反应必须在pH≤3条件下进行,且H₂O₂消耗量大而导致价格昂贵,处理成本较高等。     

Chlorella vulgaris Aldehyde Reductase Is Capable of Functioning as Ferric Reductase and of Driving the Fenton Reaction in the Presence of Free Flavin

The free flavin-dependent Fenton reaction was detected in cell-free extracts of Chlorella. The corresponding enzyme was purified to homogeneity, and its N-terminal sequence was highly homologous to those of aldo-keto reductase family enzymes. The purified enzyme displayed aldehyde reductase activity in the presence of NADPH. Additionally, it showed ferric reductase activity and drove the Fenton reaction in the presence of free FAD and NADH.     

对锰离子参与类Fenton反应机理的研究

锰主要以Mn2 形式存在,有人发现其具有与其他过渡性金属离子截然相反的抗氧化活性,采用自旋捕捉-ESR技术、芳环羟基化反应-高效液相色谱(HPLC)法和琼脂糖电泳法三种方法研究Mn2 参与类Fenton反应的情况时,均检测到Mn2 与H2O2反应产生.OH,Mn2 与H2O2反应可以发生类Fenton反应,产生.OH。这一现象的产生可能是Mn2 引起生物体内氧化损伤之故。同时显示,Mn2 的类Fenton反应是否产生.OH与反应过程Mn2 以及其他成分浓度有关(如高浓度抑制,低浓度促进),为诸多文献中Mn2 作为促氧化剂还是抗氧化剂的争论提供了可能解释。同时Mn2 能引起.OH持续低量的产生为一些慢性疾病的发生提供了合理的解释。     

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     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

从吸收光谱的变化看H2O2对豆血红蛋白的定位损伤

豆血红蛋白（Ｌｂ）是一种含铁的蛋白质,在大豆（Ｇｌｙｃｉｎｅｍａｘ）的根瘤中含量很。从新鲜根瘤中提取制备的氧合豆血红蛋白（ＬｂＯ２,含二价铁的蛋白质）是Ｌｂ的活性形式。ＬｂＯ２在可见光区５７７ｎｍ和５４０ｎｍ有吸收峰,这两个峰与ＬｂＯ２中血红素（铁卟啉）的结构密切相关;另外,ＬｂＯ２在紫外区２８０ｎｍ处还有一个吸收峰,此峰与珠蛋白（ＬｂＯ２的蛋白质部分）的构象有关．ＬｂＯ２在一定浓度的Ｈ２Ｏ２作用下,可见光的特异吸收变化迅速,而紫外吸收相对稳定,因而推测Ｈ２Ｏ２对ＬｂＯ２的损伤是有着固定的位点,这个位点是在含二价铁的卟啉环上,而不在珠蛋白中．     

Nitric oxide and Fenton/Haber-Weiss chemistry: nitric oxide is a potent antioxidant at physiological concentrations

We have examined the action of nitric oxide (NO) on the ability of Fenton's reagent (ferrous iron and hydrogen peroxide), to oxidize a number of organic optical probes. We found that NO is able to arrest the oxidation of organic compounds at concentrations of NO found in brain, in vivo. We present evidence that Fenton's reagent proceeds via a ferryl intermediate ([Fe[double bond]O]2+), before the generation of hydroxyl radical *OH. NO reacts rapidly with this ferryl, blocking the production of *OH. We propose that NO has an important role in protecting biological tissues, and the brain in particular, from Fenton chemistry.     

“Chelatable iron pool”: inositol 1,2,3-trisphosphate fulfils the conditions required to be a safe cellular iron ligand

Mammalian cells contain a pool of iron that is not strongly bound to proteins, which can be detected with fluorescent chelating probes. The cellular ligands of this biologically important “chelatable”, “labile” or “transit” iron are not known. Proposed ligands are problematic, because they are saturated by magnesium under cellular conditions and/or because they are not “safe”, i.e. they allow iron to catalyse hydroxyl radical formation. Among small cellular molecules, certain inositol phosphates (InsPs) excel at complexing Fe³⁺ in such a “safe” manner in vitro. However, we previously calculated that the most abundant InsP, inositol hexakisphosphate, cannot interact with Fe³⁺ in the presence of cellular concentrations of Mg²⁺. In this work, we study the metal complexation behaviour of inositol 1,2,3-trisphosphate [Ins(1,2,3)P ₃], a cellular constituent of unknown function and the simplest InsP to display high-affinity, “safe”, iron complexation. We report thermodynamic constants for the interaction of Ins(1,2,3)P ₃ with Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Fe²⁺ and Fe³⁺. Our calculations indicate that Ins(1,2,3)P ₃ can be expected to complex all available Fe³⁺ in a quantitative, 1:1 reaction, both in cytosol/nucleus and in acidic compartments, in which an important labile iron subpool is thought to exist. In addition, we calculate that the fluorescent iron probe calcein would strip Fe³⁺ from Ins(1,2,3)P ₃ under cellular conditions, and hence labile iron detected using this probe may include iron bound to Ins(1,2,3)P ₃. Therefore Ins(1,2,3)P ₃ is the first viable proposal for a transit iron ligand. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of Active Oxygen Radicals on Protein and Carbohydrate Moieties of Recombinant Human Erythropoietin

Our previous study showed that active oxygen radicals generated from a Fenton system and a xanthine plus xanthine oxidase system caused serious loss of in vivo bioactivity of recombinant human erythropoietin (EPO), a highly glycosylated protein. In the present study, we characterized the oxidative modifications to the protein and carbohydrate moiety of EPO, which lead to a reduction of its bioactivity. In vitro bioactivity was reduced when EPO was treated with oxygen radi cals generated from a Fenton system in the presence of 0.016 mM H₂0₂, and the reduction was directly proportional to the loss of in vivo bioactivity. SDS-PAGE analysis showed that dimer formation and degradation was observed under more severe conditions (Fenton reaction with 0.16 mM H₂0₂). The tryptophan destruction was detected at 0.016 mM H₂O₂ and well correlated with the loss of in vitro bioactivity, whereas loss of other amino acids were occurred under more severe conditions. Treatment with the Fenton system did not result in any specific damage on the carbohydrate moiety of EPO, except a reduction of sialic acid content under severe condition. These results suggest that active oxygen radicals mainly react with the protein moiety rather than the carbohydrate moiety of EPO. Destruction of tryptophan residues is the most sensitive marker of oxidative damage to EPO, suggesting the importance of tryptophan in the active EPO structure. Deglycosylation of EPO caused an increase of susceptibility to oxygen radicals compared to intact EPO. The role of oligosaccharides in EPO may be to protect the protein structure from active oxygen radicals.     

Lysozyme Modification by the Fenton Reaction and Gamma Radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H 2 O 2 ) or hydroxyl radicals produced by &#110 radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. &#110 radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met=Cys > Lys > Ile+Leu > Gly > Pro=Phe>Thr+Ala>Trp=Ser>Arg>Asp+Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met >His > Ile+Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by &#110 radiation.     

Treatment of oilfield wastewater containing polymer by the batch activated sludge reactor combined with a zerovalent iron/EDTA/air system

Laboratory-scale experiments were conducted in order to evaluate the performance of a novel treatment process for oilfield wastewater based on combining chemical oxidation, performed by a zerovalent iron (ZVI), ethylenediamine tetraacetic acid (EDTA) and air process, with biological degradation, carried out in a batch activated sludge reactor. The influence of some operating variables was studied. The results showed that the optimum pretreatment conditions were 150 mg/L EDTA, 20 g/L ZVI, and a 180-min reaction time, respectively. Under these conditions, removal efficiencies for hydrolyzed polyacrylamide (HPAM), total petroleum hydrocarbons (TPH), and chemical oxygen demand (COD) were 66%, 59%, and 45%, respectively. During the subsequent 40 h of bioremediation, the concentrations of HPAM, TPH, and COD were decreased to 10, 2 and 85 mg/L, respectively. At the end of experiments, the total removal efficiencies of HPAM, TPH, and COD were 96%, 97% and 92%, respectively.     

Iron metabolism: The low-molecular-mass iron pool

Summary This review examines various aspects of iron metabolism in mammalian and bacterial cells which support the hypothesis of the existence and the biological significance of an intracellular pool of low-molecular mass iron complexes.