Showing papers on "Hydrogen peroxide published in 2010"

Measurement of superoxide dismutase, catalase, and glutathione peroxidase in cultured cells and tissue

Abstract: Cells contain a large number of antioxidants to prevent or repair the damage caused by reactive oxygen species, as well as to regulate redox-sensitive signaling pathways. General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, whereas the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein required for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissues and cells. In general, these assays require 24–48 h to complete.

Effect of halide ions and carbonates on organic contaminant degradation by hydroxyl radical-based advanced oxidation processes in saline waters.

Abstract: Advanced oxidation processes (AOPs) generating nonselective hydroxyl radicals (HO*) provide a broad-spectrum contaminant destruction option for the decontamination of waters. Halide ions are scavengers of HO* during AOP treatment, such that treatment of saline waters would be anticipated to be ineffective. However, HO* scavenging by halides converts HO* to radical reactive halogen species (RHS) that participate in contaminant destruction but react more selectively with electron-rich organic compounds. The effects of Cl-, Br-, and carbonates (H2CO3+HCO3-+CO3(2-)) on the UV/H2O2 treatment of model compounds in saline waters were evaluated. For single target organic contaminants, the impact of these constituents on contaminant destruction rate suppression at circumneutral pH followed the order Br->carbonates>Cl-. Traces of Br- in the NaCl stock had a greater effect than Cl- itself. Kinetic modeling of phenol destruction demonstrated that RHS contributed significantly to phenol destruction, mitigating the impact of HO* scavenging. The extent of treatment efficiency reduction in the presence of halides varied dramatically among different target organic compounds. Destruction of contaminants containing electron-poor reaction centers in seawater was nearly halted, while 17beta-estradiol removal declined by only 3%. Treatment of mixtures of contaminants with each other and with natural organic matter (NOM) was evaluated. Although NOM served as an oxidant scavenger, conversion of nonselective HO* to selective radicals due to the presence of anions enhanced the efficiency of electron-rich contaminant removal in saline waters by focusing the oxidizing power of the system away from the NOM toward the target contaminant. Despite the importance of contaminant oxidation by halogen radicals, the formation of halogenated byproducts was minimal.

Mechanistic investigation into antibacterial behaviour of suspensions of ZnO nanoparticles against E. coli

Abstract: Aqueous suspensions containing 4.45 × 10−5 − 1.25 × 10−3 M ZnO particles exhibit a strong antibacterial activity against E. coli under the dark conditions. The dominant mechanisms of such antibacterial behaviour are found to be either or both of chemical interactions between hydrogen peroxide and membrane proteins, and chemical interactions between other unknown chemical species generated due to the presence of ZnO particles with the lipid bilayer. The effect of direct physical interactions between nanoparticles and biological cells are found to play a relatively small role under the conditions of this study.

Methods for detection and measurement of hydrogen peroxide inside and outside of cells

Abstract: Hydrogen peroxide (H2O2) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H2O2 requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H2O2 in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3’5’-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H2O2, methods based on dihydro compounds such as 2’,7’-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H2O2. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H2O2-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H2O2 in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H2O2 can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H2O2, they cannot be used to monitor transient changes in H2O2 concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H2O2 and can be targeted to various compartments of the cell, but they are not selective for H2O2 because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H2O2-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.

Determination and Detection of Reactive Oxygen Species (ROS), Lipid Peroxidation, and Electrolyte Leakage in Plants

Abstract: Reactive oxygen species or intermediates are formed by the incomplete reduction of oxygen. Organisms living in aerobic environment generate various kinds of reactive oxygen species (ROS) molecules, such as superoxide (*O(2)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radical (OH(-)), singlet oxygen, and lipid hydroperoxides. ROS are highly reactive molecules and are extremely unstable, so detection of ROS relies on measuring the end products that are formed when they react with particular substances. The end products can be measured by changes in their fluorescence, color, or luminescence. ROS causes lipid peroxidation wherein the lipids in the cell membranes are damaged. Lipid peroxidation is usually quantified using a colorimetric assay. When ROS concentrations reach a certain threshold, it activates a programmed cell death response in the cells. This is quantified by measuring the amount of ion leakage. ROS such as superoxide and hydrogen peroxide have been detected traditionally by staining techniques. Superoxide anion is detected with nitroblue tetrazolium (NBT) and hydrogen peroxide by Diaminobenzidine tetrahydrochloride (DAB) staining. In this chapter, methods for determining total ROS and lipid peroxidation assay, histochemical staining techniques for superoxide and H(2)O(2) molecules are described.

Mode of action of hydrogen peroxide and other oxidizing agents: differences between liquid and gas forms

Abstract: Objectives; Antimicrobials such as chlorine dioxide, peracetic acid and hydrogen peroxide (H2O2) share a basic mechanism of action (chemical oxidation of cellular components), but profound differences arise in their efficacy against microorganisms. Optimization of activity requires an understanding of their interaction with microbial targets and a clear differentiation between the chemical efficacies of each oxidative biocide. This study aimed to elucidate the biochemical mechanisms of action of oxidizing biocides at a macromolecular level, using amino acids, protein and an enzyme as model substrates for the action of each biocide. Methods: The interactions of a number of oxidising agents (liquid and gaseous H2O2, ClO2, peracetic acid formulations) with amino acids, proteins (bovine serum albumin and aldolase) and enzymes were investigated by spectrophotometry, SDS-PAGE and alkaline phosphatase activity measurements. Results: Biocide reactions yielded different types of oxidative structural change and different degrees of oxidation to amino acids and proteins, and differences in activity against a microbial enzyme. In particular there was a marked difference in the interactions of liquid H2O2 and gaseous H2O2 with the macromolecules, the latter causing greater oxidation; these results explain the dramatic differences in antimicrobial efficacy between liquid and gas peroxide. Conclusions: These results provide a comprehensive understanding of the differences in interactions between a number of oxidizing agents and macromolecules commonly found in microbial cells. Biochemical mechanistic differences between these oxidative biocides do exist and lead to differential effects on macromolecules. This in turn could provide an explanation as to their differences in biocidal activity, particularly between liquid and gas peroxide.

Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity

Abstract: The free radical theory of aging posits oxidative damage to macromolecules as a primary determinant of lifespan. Recent studies challenge this theory by demonstrating that in some cases, longevity is enhanced by inactivation of oxidative stress defenses or is correlated with increased, rather than decreased reactive oxygen species and oxidative damage. Here we show that, in Saccharomyces cerevisiae, caloric restriction or inactivation of catalases extends chronological lifespan by inducing elevated levels of the reactive oxygen species hydrogen peroxide, which activate superoxide dismutases that inhibit the accumulation of superoxide anions. Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases.

In situ liquid phase synthesis of hydrogen peroxide from molecular oxygen using gold nanoparticle-loaded titanium(IV) dioxide photocatalyst.

Abstract: Gold nanoparticle loading has led to a drastic enhancement of TiO2-photocatalized generation of H2O2 from O2 with a unique inversed volcano-type relation between the activity and Au particle size.

Direct Synthesis of Hydrogen Peroxide and Benzyl Alcohol Oxidation Using Au−Pd Catalysts Prepared by Sol Immobilization†

Abstract: We report the preparation of Au-Pd nanocrystalline catalysts supported on activated carbon prepared via a sol-immobilization technique and explore their use for the direct synthesis of hydrogen peroxide and the oxidation of benzyl alcohol. In particular, we examine the synthesis of a systematic set of Au-Pd colloidal nanoparticles having a range of Au/Pd ratios. The catalysts have been structurally characterized using a combination of UV-visible spectroscopy, transmission electron microscopy, STEM HAADF/XEDS, and X-ray photoelectron spectroscopy. The Au-Pd nanoparticles are found in the majority of cases to be homogeneous alloys, although some variation is observed in the AuPd composition at high Pd/Au ratios. The optimum performance for the synthesis of hydrogen peroxide is observed for a catalyst having a Au/Pd 1:2 molar ratio. However, the competing hydrogenation reaction of hydrogen peroxide increases with increasing Pd content, although Pd alone is less effective than when Au is also present. Investigation of the oxidation of benzyl alcohol using these materials also shows that the optimum selective oxidation to the aldehyde occurs for the Au/Pd 1:2 molar ratio catalyst. These measured activity trends are discussed in terms of the structure and composition of the supported Au-Pd nanoparticles.

Voltammetric Detection of Hydrogen Peroxide at Carbon Fiber Microelectrodes

Abstract: Hydrogen peroxide is a reactive oxygen species that is implicated in a number of neurological disease states and that serves a critical role in normal cell function. It is commonly exploited as a reporter molecule enabling the electrochemical detection of nonelectroactive molecules at electrodes modified with substrate-specific oxidative enzymes. We present the first voltammetric characterization of rapid hydrogen peroxide fluctuations at an uncoated carbon fiber microelectrode, demonstrating unprecedented chemical and spatial resolution. The carbon surface was electrochemically conditioned on the anodic scan and the irreversible oxidation of peroxide was detected on the cathodic scan. The oxidation potential was dependent on scan rate, occurring at +1.2 V versus Ag/AgCl at a scan rate of 400 V.s(-1). The relationship between peak oxidation current and concentration was linear across the physiological range tested, with deviation from linearity above 2 mM and a detection limit of 2 muM. Peroxide was distinguished from multiple interferents, both in vitro and in brain slices. The enzymatic degradation of peroxide was monitored, as was peroxide evolution in response to glucose at a glucose oxidase modified carbon fiber electrode. This novel approach provides the requisite sensitivity, selectivity, spatial and temporal resolution to study dynamic peroxide fluctuations in discrete biological locations.

Gold on Diamond Nanoparticles as a Highly Efficient Fenton Catalyst

Abstract: The Fenton reaction consists of the generation of highly aggressive hydroxyl radicals from hydrogen peroxide and is widely used to degrade organic pollutants. Due to its general applicability, the Fenton reaction is employed in water and soil disinfection/remediation and for removal of non-biodegradable chemicals. The main limitation of the Fenton reaction is the consumption of stoichiometric amounts of transition metals, mostly iron. There is considerable incentive in developing a catalytic Fenton process using exclusively hydrogen peroxide and a catalyst. Herein we report that gold nanoparticles grafted on nanoparticulate diamond catalyze the formation of hydroxyl radicals from hydrogen peroxide with at least 79 % efficiency and reach a turnover number of 321 000, many orders of magnitude higher than any currently available catalysts. This extraordinary activity is derived directly from the nanometric diameters of gold and diamond (“nanojewels”) and from the remarkable inertness of the diamond surface. The Fenton reaction, in which highly aggressive hydroxyl radicals (HOC) are generated from H2O2 by reduction with Fe II ,C u II , or other transition metal salts, is a general process that can be used for the degradation/mineralization of recalcitrant organic pollutants as well as for disinfection. [1–4] In spite of the wide applicability of the Fenton reaction for decomposing almost any organic compound, its widespread use for pollution abatement and disinfection is limited by the need for stoichiometric amounts of Fe II or other transition metals. Most of the efforts to transform the Fenton reaction from a stoichiometric to a catalytic process have met with failure or at best can produce HOC with remarkably low efficiency. [5] For instance, the photo-Fenton process requires transparency of the solution (a prerequisite not frequently fulfilled in polluted waters or soils) and consumes “expensive” photons as stoichiometric reagents. A large number of iron-containing solids such as iron-exchanged zeolites and montmorillonites have also been reported as heterogeneous

Peroxiredoxin IV protects cells from oxidative stress by removing H2O2 produced during disulphide formation

Abstract: Disulphide formation within the endoplasmic reticulum (ER) requires the activity of the ER oxidase Ero1, and as a consequence, generates hydrogen peroxide. The production of hydrogen peroxide is thought to lead to oxidative stress that ultimately results in apoptosis. Here, we show that mammalian peroxiredoxin IV (PrxIV) metabolises hydrogen peroxide produced by Ero1. We demonstrate that the presence of PrxIV within the ER provides a cytoprotective effect against stresses known to enhance Ero1 activity and perturb ER redox balance. Increased Ero1 activity and production of hydrogen peroxide led to preferential hyperoxidation of PrxIV relative to peroxiredoxins in other cellular compartments. The hyperoxidation was increased by the upregulation of Ero1 and by the expression of a hyperactive Ero1. These findings provide the first evidence for an enzymatic mechanism that facilitates peroxide removal from the ER, and show that the oxidation status of PrxIV acts as a marker for ER oxidative stress.

Role of metabolically generated reactive oxygen species for lipotoxicity in pancreatic β‐cells

Abstract: Chronically elevated concentrations of non-esterified fatty acids (NEFAs) in type 2 diabetes may be involved in β-cell dysfunction and apoptosis It has been shown that long-chain saturated NEFAs exhibit a strong cytotoxic effect upon insulin-producing cells, while short-chain as well as unsaturated NEFAs are well tolerated Moreover, long-chain unsaturated NEFAs counteract the toxicity of palmitic acid Reactive oxygen species (ROS) formation and gene expression analyses together with viability assays in different β-cell lines showed that the G-protein-coupled receptors 40 and 120 do not mediate lipotoxicity This is independent from the role, which these receptors, specifically GPR40, play in the potentiation of glucose-induced insulin secretion by saturated and unsaturated long-chain NEFAs Long-chain NEFAs are not only metabolized in the mitochondria but also in peroxisomes In contrast to mitochondrial β-oxidation, the acyl-coenzyme A (CoA) oxidases in the peroxisomes form hydrogen peroxide and not reducing equivalents As β-cells almost completely lack catalase, they are exceptionally vulnerable to hydrogen peroxide generated in peroxisomes ROS generation in the respiratory chain is less important because overexpression of catalase and superoxide dismutase in the mitochondria do not provide protection Thus, peroxisomally generated hydrogen peroxide is the likely ROS that causes pancreatic β-cell dysfunction and ultimately β-cell death

Photolysis of Hydrogen Peroxide, an Effective Disinfection System via Hydroxyl Radical Formation

Abstract: The relationship between the amount of hydroxyl radicals generated by photolysis of H(2)O(2) and bactericidal activity was examined. H(2)O(2) (1 M) was irradiated with laser light at a wavelength of 405 nm to generate hydroxyl radicals. Electron spin resonance spin trapping analysis showed that the amount of hydroxyl radicals produced increased with the irradiation time. Four species of pathogenic oral bacteria, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Enterococcus faecalis, were used in the bactericidal assay. S. mutans in a model biofilm was also examined. Laser irradiation of suspensions in 1 M H(2)O(2) resulted in a >99.99% reduction of the viable counts of each of the test species within 3 min of treatment. Treatment of S. mutans in a biofilm resulted in a >99.999% reduction of viable counts within 3 min. Other results demonstrated that the bactericidal activity was dependent on the amount of hydroxyl radicals generated. Treatment of bacteria with 200 to 300 μM hydroxyl radicals would result in reductions of viable counts of >99.99%.