Killing of bacillus spores by aqueous dissolved oxygen,ascorbic acid,and copper ions

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu(2+)) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O(2) is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.     

Reactions of Low Valent Transition Metal Complexes with Hydrogen Peroxide. Are they “Fenton-Like” or not? 4. The Case of Fe(II)L,L = Edta; Hedta and Tcma

The question whether hydroxyl free radicals are formed in the reactions of divalent iron complexes Fe(II)L; L = edta; hedta; tcma (tcma = l-acetato-l,4,7-triazacyclononane) with hydrogen peroxide in neutral and slightly acidic solutions was studied by using the β elimination reaction as an assay for the formation of hydroxyl free radicals, OH. The results show that at pH<5.5 the iron(II)peroxide intermediate complex decomposes rapidly to yield free hydroxyl radicals for L=edta and hedta. This is in contrast to the mechanism of the corresponding Fe(II)nta peroxide complex, which probably decomposes to form Fe(IV)nta which then reacts with organic substrates to yield aliphatic free radicals. Thus, the non-participating ligand L has an appreciable effect on the mechanism of reaction of the metal center with hydrogen peroxide. Blank experiments using ionizing radiation as the source of CH₂CR(CH₃)OH, R = H or CH₃ radicals indicate that when L = tcma intermediates of the type LFe^III-CH₂CR(CH₃)OH_aq are formed, but their major mode of decomposition is not the β elimination reaction. Thus, the present assay for the formation of hydroxyl free radicals by the Fenton Reaction does not fit the latter system.     

Presence of Hydrogen Peroxide,a Source of Hydroxyl Radicals,in Acid Electrolyzed Water

Background

Acid electrolyzed water (AEW), which is produced through the electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution, is used as a disinfectant in various fields because of its potent antimicrobial activity. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity.

Methodology/Principal Findings

The aim of the present study is to detect hydroxyl radicals in AEW. The hydroxyl radicals in AEW prepared under different conditions were determined using an electron spin resonance (ESR) technique. A signal from 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH, an adduct of DMPO and the hydroxyl radical, was detected in AEW prepared by double or triple electrolyses of 1% NaCl but not of 0.1% NaCl solution. Then the presence of hydrogen peroxide as a proposed source of hydroxyl radicals was examined using a combination of ESR and a Fenton reaction. The DMPO-OH signal was clearly detected, even in AEW prepared by single electrolysis of 0.1% NaCl solution, when ferrous sulfate was added to induce a Fenton reaction, indicating the presence of hydrogen peroxide in the AEW. Since sodium formate, a hydroxyl radical scavenger, did not affect the bactericidal activity of AEW, it is concluded that the radical is unlikely to contribute to the antimicrobial activity of AEW, although a small amount of the radical is produced from hydrogen peroxide. Dimethyl sulfoxide, the other hydroxyl radical scavenger used in the present study, canceled the bactericidal activity of AEW, accompanied by complete depletion of free available chlorine, suggesting that hypochlorous acid is probably a major contributor to the antimicrobial activity.

Conclusions

It is strongly suggested that although hydrogen peroxide is present in AEW as a source of hydroxyl radicals, the antimicrobial activity of AEW does not depend on these radicals.     

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.     

Covalently bound fluorescent probes as reporters for hydroxyl radical penetration into liposomal membranes

The ability of hydroxyl radicals to penetrate into liposomal model membranes (dimyristoylphosphatidylcholine) has been demonstrated. Liposomes were prepared and then characterized by digital fluorescence microscopy and dynamic light scattering after extrusion to determine liposomal lamellarity, size, and shape. Hydroxyl radicals were generated in the surrounding aqueous medium using a modified Fenton reagent (hydrogen peroxide and Fe²⁺) with the water-soluble iron chelator EDTA. High and low doses of radical were used, and the low dose was achieved with physiologically relevant iron and peroxide concentrations. Fluorescent probes covalently bound to the membrane phospholipid were used, including two lipophilic pyrenyl probes within the membrane bilayer and one polar probe at the water–membrane interface. Radical reactions with the probes were monitored by following the decrease in fluorescence and by observing oxidation products via matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Differences in the probe position within the membrane were correlated with the reactivity of the probe to assess radical access to the site of the probe. For all probes, reaction rates increased with increasing temperature. Within the membrane bilayer, reaction rates were greater for the probe closest to the membrane–water interface. Cholesterol protected these probes from oxidation. Kinetic models, scavenger studies, and product identification studies indicated that hydroxyl radical reacted directly with the in-membrane probes without the mediation of a secondary radical.     

Abatement of Sorbed Crude Oil by Heterogeneous Fenton Process Using A Contaminated Soil Pre-Impregnated with Dissolved Fe(II) and Humic Acid

Dissolved Fe(II) and humic acid (HA) were pre-impregnated into contaminated soil to catalyze hydrogen peroxide to remove crude oil (CO). The effects of parameters such as initial Fe(II), HA and H₂O₂ concentrations on the oxidation of total petroleum hydrocarbon (TPH) were investigated using response surface methodology based on Box–Behnken design. The rate of hydrogen peroxide decomposition is decreased by pre-impregnating with dissolved Fe(II) + HA compared with only pre-impregnated Fe(II) and modified Fenton (MF). Oxygen evolution is the predominant route of hydrogen peroxide decomposition at natural pH. Unlike O₂ evolution, the kinetics of hydroxyl radical (OH^?) production are clearly uncoupled from H₂O₂ decay in these systems. The steady-state hydroxyl radical production rate is higher in the systems with pre-impregnated dissolved Fe(II) and HA, and more significance is the decrease in detectable TPH (70.84% removal efficiency) when soil is pre-impregnated with dissolved 25 mM Fe(II) + 0.7 mg/mL HA, and with the application of 700 mM H₂O₂, possibly due to hydrogen peroxide catalyzed by the iron of this complex (CO-HA–Fe(II)) producing hydroxyl radical in close proximity to the CO. Meanwhile, the removal efficiency of C₂₁–C₃₀ is up to 65.69%, which is 2.6 times higher than that of the MF (25.52%).     

Homogeneous butyrylation and lauroylation of poplar wood in the ionic liquid 1-butyl-3-methylimidazolium chloride

Wood meal was dissolved under identical conditions in the ionic liquid 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) and homogeneously esterified with butyryl chloride and lauroyl chloride in the presence of triethylamine as a neutralizer. The effect of the molar ratio of reagent to the hydroxyl groups in wood on the properties of the esterified wood and the possible mechanism of the homogeneous esterification of wood in this reaction medium were studied. Furthermore, the physicochemical properties of the esterified wood were characterized by FTIR, solid-state CP/MAS ¹³C NMR, TGA/DTG and SEM. The results confirmed that the homogeneous modifications were successfully processed and highly substituted wood esters could be obtained by reacting the dissolved wood in this homogeneous system.     

Mechanisms for survival of bacteria in oxidative stress and iron ions deficiency

The influence of culture medium Fe2+ content on the resistance of Escherichia coli to hydroxyl radicals formed in the presence of Fe2+ and hydrogen peroxide in Fenton reaction was investigated. It was founded that a lack of Fe2+ in a culture medium increased resistance of bacteria to hydroxyl radicals but not to hydrogen peroxide. The suggestion was made that the lack of Fe2+ starts up synthesis of metabolites which inactivate hydroxyl radical or block Fe2+ ions participating in Fenton reaction. The phenomenon under study is considered to be a possible mechanism for survival of bacteria in oxidative stress and iron ions deficiency.     

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.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Benzyl viologen-mediated in vivo and in vitro inactivation of glutamine synthetase in Azotobacter chroococcum

Summary Addition of benzyl viologen to a cell suspension of the aerobic bacterium Azotobacter chroococcum growing on nitrate resulted in a rapid loss of glutamine synthetase activity as assayed in situ. When a glutamine synthetase preparation which exhibited NADH-benzyl viologen oxidoreductase activity was incubated, under air, with NADH and benzyl viologen, glutamine synthetase was inactivated in a short period of time. This in-vitro inactivation process could be prevented in the presence of added catalase, thus indicating that hydrogen peroxide was involved in the process, and by EDTA, suggesting that metal ions are also involved. The characteristics of the benzyl viologen-dependent glutamine synthetase inactivation observed with externally added H₂O₂ and a preincubated sample are similar.Inhibition of glutamine synthetase inactivation by histidine suggests that hydroxyl radicals, or something with similar reactivity, is the inactivating agent. The fact that inactivation can also be catalyzed by a model system consisting of Fe²⁺ and H₂O₂ leads to the conclusion that hydroxyl radicals are most likely produced in a Fenton reaction in which hydrogen peroxide reacts with adventitious iron ions.Since A. chroococcum contained a high level of catalase it may be concluded that cellular compartmentation plays an important role in the in-vivo inactivation of glutamine synthetase.     

Spin Trapping of Ibuprofen Radicals: Evidence That Ibuprofen is a Hydroxyl Radical Scavenger

The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2–(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vim. Ibuprofen (IBU; 0.1–50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5–2.0mM). and either 5.5-dimethyl-l-pyrroline-N-oxide (DMPO; 40mM) or a-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (l mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (a_N = 16.1G, a^H_β = 24.0G) and PBN (a_N = 15.7G. a^H_β = 4.4G and a_N = 17.0G, a^H_β = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (l mM), hydrogen peroxide (lmM), PBN (48mM), and increasing IBU concentrations. Therefore. we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hydroxyl radicals would be generated, less IBU radicals formed and the signal height of IBU radicals trapped by PBN would have decreased. However, these data do not fully exclude the possiblity that IBU may, to some extent. also chelate iron. Scavenging of hydroxyl radicals may be one of the mechanisms responsible for the beneficial action of IBU during the management of several rheumatic diseases. However, the IBU radicals produced when IBU scavenges hydroxyl radicals are reactive. and may be associated with the reported toxicity of this therapeutic agent.     

The influence of pH on OH scavenger inhibition of damage to deoxyribose by Fenton reaction

Summary Hydroxyl radicals (OH') can be formed in aqueous solution by direct reaction of hydrogen peroxide (H₂O₂) with ferrous salt (Fenton reaction). OH' damage to deoxyribose, measured as formation of thiobarbituric acid-reactive material, was evaluated at different pHs to study the mechanism of action of classical OH' scavengers. OH' scavenger effect on Fe²⁺ oxidation was also evaluated in the same experimental conditions. In the absence of OH' scavengers, OH' damage to deoxyribose is higher at acidic compared to neutral and moderately basic pH. At acidic pH deoxiribose is per se able to inhibit Fe²⁺ oxidation by H₂0₂. Most of OH' scavengers tested inhibit deoxyribose damage and Fe²⁺ oxidation in a similar manner: both inhibitions are most relevant at acidic pH and decrease by increasing the pH. These results are not due to OH' scavenger inhibition of Fenton reaction. The influence of pH on the parameters studied appears to be due to the competition of deoxyribose and OH' scavengers for iron. These results suggest the prominent role of iron binding in the degradation of deoxyribose and in the OH' scavenging ability of different compounds. Results obtained with triethylenetetramine, a iron chelator with a low rate constant with OH', confirm that both deoxyribose and the OH' scavengers interact with iron bringing about a site specific Fenton reaction; that the OH' formed at these sites oxidize these molecules to their radical forms which in turn reduce the Fe^3– produced by Fenton reaction. The results presented indicate that most of classical OH' scavengers exert their effect predominantly by preventing the site specific reaction between Fe²⁺ and H₂0₂ on the deoxyribose molecule.     

Terephthalic acid: A dosimeter for the detection of hydroxyl radicals in vitro

Hydroxylation reactions of aromatic compounds have been used to detect hydroxyl radicals produced by gamma irradiation and ultrasound. The present study investigated the suitability of terephthalic acid (THA) as a hydroxyl radical dosimeter for general use in biologically relevant reactions. Hydroxyl radicals were generated by: (1) irradiating, THA with a 254 nm ultraviolet; (2) irradiating with gamma rays from a cesium source; and (3) generating hydroxyl radicals with 1 mM H₂O₂ and 10 μM Cu⁺². In each of the three experiments, a fluorescent product was generated which exhibited identical fluorescent excitation and emission spectra. THA is non-fluorescent, eliminating the problem of a high initial background. Because THA has four ring hydrogens, only one mon-hydroxylated isomer was formed. The hydrogen peroxide reaction was dependent on the presence of a metal and cupric ions were effective in enhancing the reaction. With a Cu⁺² concentration of 10 μM, the reation was linear between 0–30 mM H₂O₂. Catalase abolished the reaction at a concentration of 100 μg/ml and the effects could still be observed at 10 ng/ml, consistent with the very high rate at which catalase destroys hydrogen peroxide. Tertbutyl- hydroperoxide did not generate any fluorescence in this system which makes THA a very specific detector of hydroxyl radicals.     

The production of oxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: An electron paramagnetic resonance study of the response to phorbol myristate acetate

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.     

A hypothesis for thein vivo antioxidant action of salicyclic acid

This paper presents a new hypothesis for the physiological antioxidant action of salicylate. Current theories have focused on the radical scavenging nature of salicylate. This explanation may have limitations because it is unlikely that salicylate reaches the necessary concentrations to effectively prevent damage to cell components. We propose that salicylic acid decreases the flux of hydroxyl radicals through chelation, which causes a redox deactivation mechanism of iron Fenton reaction centers. This is due to voltammetric results which indicate that the iron-salicylate complex does not have the thermodynamic driving force to act as an effective Fenton reagent necessary for the production of damaging oxygen-containing radicals. Furthermore, despite the more facile thermodynamics associated with Fenton-type processes at acidic pH values, the complex maintains Fenton inactivity due to a pH-sensitive redox potential shift that follows asE _Fe[Sal] = 0.793 - (0.059 pH). This is important since inflammation sites are acidic relative to healthy tissue. This redox potential shift is unique to salicylates when compared with other common iron chelation agents such as EDTA. Further evidence for the lack of Fenton-type reactivity of the iron-salicylate complex is offered in the form of oxidation studies of calf thymus (CT) DNA by hydrogen peroxide. Salicylate prevents the iron-catalyzed oxidation of CT-DNA strands as indicated by the detection of the constituent bases by HPLC. However, salicylates were not able to prevent the copper-catalyzed oxidation of CT-DNA. These results are predicted by the cyclic voltammetry of copper-salicylate, which confirms that it is an effective Fenton-type catalyst, further adding to the proof that salicylate acts by redox deactivation of iron, not by hydroxyl radical scavenging. Finally, the iron-salicylate e.m.f. suggests that it may also act as a superoxide dismutase, which indicates another possible important antioxidant feature.     

Activation mechanism for N-nitroso-N-methylbutylamine mutagenicity by radical species

N-Nitrosodialkylamines are known to be potent indirect-acting mutagens/carcinogens, which are activated by cytochrome P450. The reaction product of N-nitroso-N-methylbutylamine (NMB) with modified Fenton’s reagent supplemented with copper salt (Fe²⁺–Cu²⁺–H₂O₂) was reported to be mutagenic in Salmonella typhimurium TA1535 without S9 mix. In this study, the NMB activation mechanism was investigated by ESR spectroscopy with radical trapping agents to detect radical species and also by observing changes in mutagenic potency with a Salmonella strain in the Ames assay in the presence of radical trapping agents. In ESR spectroscopy experiments, the hydroxyl radical generated from the modified Fenton’s reagent was detected using the hydroxyl radical trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Since the amount of the DMPO–OH adduct decreased with the addition of NMB, hydroxyl radical was presumed to react with NMB followed by the generation of nitric oxide (NO), which was detected as CarboxyPTI through reaction with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (CarboxyPTIO). The mutagenicity of the reaction extract decreased following the addition of DMPO or CarboxyPTIO. Furthermore, the mutagenicity of the reaction product in the presence of DMPO was enhanced by the addition of NO. The reaction product from NMB with Fe²⁺–Cu²⁺–NO in the absence of H₂O₂ was mutagenic, and this activity increased with the introduction of additional NO. These findings suggest that hydroxyl radical takes part in the generation of NO from NMB and that NO plays an important role in NMB activation in the presence of Fe²⁺ and Cu²⁺.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Coupled oxidation of NADPH with thiols at neutral pH.

NADPH and NADH are rapidly oxidized in neutral imidazole chloride buffer at 30 °C in the presence of mercaptoethanol or dithiothreitol. The product of the NADPH reaction has been determined to be enzymically active NADP⁺. Oxidation of the pyridine nucleotides is coupled to the autooxidation of the thiol and is inhibited by ethylenediamine tetraacetic acid, stimulated by metal ions (FeSO₄), and requires oxygen. The rapid oxidation of thiols and NADPH at neutral pH was found to occur only in imidazole and, to a lesser extent, in histidine buffer. Under the conditions employed, 300 μm dithiothreitol and 30 μm NADPH are oxidized in 30 min. Both NADPH and thiol oxidations are inhibited by catalase, whereas superoxide dismutase only inhibits the oxidation of NADPH. NADPH oxidation is also inhibited by the hydroxyl radical scavengers formate, mannitol, or benzoate. A reaction mechanism is proposed in which imidazole promotes the metal-catalyzed oxidation of thiols at neutral pH. The superoxide radical generated either by the thiol oxidation or directly oxidizes NADPH or forms hydrogen peroxide and hydroxyl radicals which can oxidize NADPH. Hydrogen peroxide is also involved in the autooxidation of the thiol.