Detection of hydrogen peroxide with Amplex Red: interference by NADH and reduced glutathione auto-oxidation

We report here that reduced pyridine nucleotides and reduced glutathione result in an oxidation of Amplex Red by dioxygen that is dependent on the presence of horseradish peroxidase (HRP). Concentrations of NADH and glutathione typically found in biological systems result in the oxidation of Amplex Red at a rate comparable to that produced, for example, by respiring mitochondria. The effects of NADH and glutathione in this assay system are likely to be the result of H(2)O(2) generation via a superoxide intermediate because both catalase and superoxide dismutase prevent the oxidation of Amplex Red. These results suggest caution in the assay of H(2)O(2) production in biological systems using the Amplex Red/HRP because the assay will also report the mobilization of NADH or glutathione. However, the interruption of this process by the addition of superoxide dismutase offers a simple and reliable method for establishing the source of the oxidant signal.     

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder's reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinder's reagent demonstrated applicability to other analyte biosensing tools.     

Thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) from Lenzites betulinus

A thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) was purified from the culture medium of Lenzites betulinus by ion exchange chromatography, gel filtration and isoelectric focusing chromatography. The MnP purified from L. betulinus (L-MnP) has a molecular mass of 40 kDa and its isoelectric point was determined to be 6.2. The first 19 amino acids at the N-terminal end of the L-MnP sequence were found to exhibit 74% identity with those of a Phlebia radiata MnP. L-MnP was proved to have the highest hydrogen peroxide tolerance among MnPs reported so far. It retained more than 60% of the initial activity after thermal treatment at 60°C for 60 min, and also retained more than 60% of the initial activity after exposure to 10 mM hydrogen peroxide for 5 min at 37°C.     

Peroxidase-catalysed oxidation of chlorophyll by hydrogen peroxide

Chlorophyll is effectively bleached by H₂O₂ in the presence of certain phenols and peroxidase (EC 1.11.1.7) extracted from acetone powders of orange flavedo (Citrus sinensis). Optimal conditions for chlorophyll: hydrogen peroxide oxidoreductase include: pH, 5.9; [H₂O₂] 222 μM; ionic strength 0.11. A phenol is required and resorcinol is the most effective. Catechol and hydroquinone are inhibitory. Chlorophyll a, chlorophyllide a, and chlorophyll b all have similar V_max but K_m for chlorophyll a is about one-third that of chlorophyll b, while the K_m for chlorophyllide a is about one-half that of chlorophyll a. Pheophytin a was much less reactive than chlorophyll a, and Mg²⁺ included in the reaction system did not affect rates of pheophytin destruction.     

The effect of different antioxidants on glutathione turnover in human cell lines and their interaction with hydrogen peroxide

BACKGROUND: Glutathione plays crucial roles in antioxidant defence and glutathione deficiency contributes to oxidative stress and may therefore play a key role in the pathogenesis of many diseases. The objectives of the present study were to evaluate the effects on glutathione turnover of thiol and non-thiol antioxidants in human cell cultures and if any of the antioxidant had a short-term cellular effect against different levels of hydrogen peroxide. METHODS: We have investigated the effect on the total glutathione amount in HeLa and hepatoma cell cultures of thiol antioxidants in comparison with non-thiol antioxidants, such as a copper chelator, Vitamin C, and a flavonoid. Furthermore, we have investigated the short-term (within 24h) interaction of the different antioxidants with hydrogen peroxide. RESULTS AND CONCLUSION: Lipoic acid and quercetin (Quer) were the two antioxidants that showed the highest stimulation of glutathione synthesis in cell cultures as judged by the total glutathione amount. However, no antioxidant protected against hydrogen peroxide present in concentrations that lowered cell protein. This finding may be attributed to the fact that it is necessary to incubate cell cultures with antioxidants or small doses of oxidants for a period before the cultures are exposed to hydrogen peroxide in order to enhance the antioxidant defence. The presence of Quer and Vitamin C lowered cell protein and total glutathione even in cell cultures containing hydrogen peroxide in concentrations that did not lower cell protein. This finding might be attributed to pro-oxidant properties and formation of excess reactive oxygen species in the presence of Quer and Vitamin C.     

Vanadium catalyzed oxidation with hydrogen peroxide

Vanadium peroxides are known as very effective oxidants of different organic and inorganic substrates. In this short account reactivity, structural and mechanistic studies concerning the behaviour of peroxovanadates toward a number of different substrates are collected. Homogeneous and two-phase systems are presented, in addition, interesting synthetic results obtained with the use of ionic liquids as reaction media are also presented.     

Ascorbate peroxidase, a scavenger of hydrogen peroxide in glyoxysomal membranes

Efficient destruction of hydrogen peroxide (H(2)O(2)) in peroxisomes requires the action of an anti-oxidant defense system, which consists of low molecular weight anti-oxidant compounds, such as ascorbic acid, along with protective enzymes, such as catalase and ascorbate peroxidase (APX). We investigated the contribution of the ascorbate enzyme system to the consumptions of H(2)O(2) and NADH within glyoxysomes of germinating castor beans (Ricinus communis). We solubilized the glyoxysomal membrane APX (gmAPX) using octyl-glucoside and purified its activity by gel filtration. The activity was associated with a 34kDa protein, as determined by SDS-gel electrophoresis and Western blotting. The enzymatic properties of gmAPX were studied and this enzyme was found to utilize ascorbic acid as its most effective natural electron donor but it would also use pyrogallol and guaiacol at a smaller extent. Cyanide and azide drastically inhibited gmAPX, as well as certain thiol-modifying reagents and some metal chelators. The inhibition by cyanide and azide of the enzyme combined with its absorption spectra confirmed that it is a hemoprotein. The apparent K(m) value of the enzyme for ascorbic acid was 300 microM while the K(m) for H(2)O(2) was 60 microM. APX in the glyoxysomal membrane can work in cooperation with monodehydroascorbate reductase to oxidize NADH, regenerate ascorbate, detoxify H(2)O(2), and protect the integrity of glyoxysomal proteins and membranes.     

Determination of hydrogen peroxide generated by reduced nicotinamide adenine dinucleotide oxidase

Hydrogen peroxide can be conveniently determined using horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). However, interference occurs among assay components in the presence of reduced nicotinamide adenine dinucleotide (NADH) that is also a substrate of NADH oxidase. So, depletion of NADH is required before using the HRP method. Here, we report simple and rapid procedures to accurately determine hydrogen peroxide generated by NADH oxidase. All procedures developed were based on the extreme acid lability of NADH and the stability of hydrogen peroxide, because NADH was decomposed at pH 2.0 or 3.0 for 10 min, while hydrogen peroxide was stable at pH 2.0 or 3.0 for at least 60 min. Acidification and neutralization were carried out by adjusting sample containing NADH up to 30 microM to pH 2.0 for 10 min before neutralizing it back to pH 7.0. Then, hydrogen peroxide in the sample was measured using the HRP method and its determination limit was found to be about 0.3 microM. Alternatively, hydrogen peroxide in samples containing NADH up to 100 microM could be quantitated using a modified HRP method that required an acidification step only, which was found to have a determination limit of about 3 microM hydrogen peroxide in original samples.     

Reaction of ferrous lactoperoxidase with hydrogen peroxide and dioxygen: an anaerobic stopped-flow study

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears, and saliva and has physiological significance in antimicrobial defense which involves (pseudo-)halide oxidation. LPO compound III (a ferrous-dioxygen complex) is known to be formed rapidly by an excess of hydrogen peroxide and could participate in the observed catalase-like activity of LPO. The present anaerobic stopped-flow kinetic analysis was performed in order to elucidate the catalytic mechanism of LPO and the kinetics of compound III formation by probing the reactivity of ferrous LPO with hydrogen peroxide and molecular oxygen. It is shown that ferrous LPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl LPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (7.2+/-0.3) x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C. The H2O2-mediated conversion of compound II to compound III follows also second-order kinetics (220 M(-1) s(-1) at pH 7.0 and 25 degrees C). Alternatively, compound III is also formed by dioxygen binding to ferrous LPO at an apparent bimolecular rate constant of (1.8+/-0.2) x 10(5) M(-1) s(-1). Dioxygen binding is reversible and at pH 7.0 the dissociation constant (K(D)) of the oxyferrous form is 6 microM. The rate constant of dioxygen dissociation from compound III is higher than conversion of compound III to ferric LPO, which is not affected by the oxygen concentration and follows a biphasic kinetics. A reaction cycle including the redox intermediates compound II, compound III, and ferrous LPO is proposed, which explains the observed (pseudo-)catalase activity of LPO in the absence of one-electron donors. The relevance of these findings in LPO catalysis is discussed.     

Melatonin does not "directly scavenge hydrogen peroxide": demise of another myth

We have reexamined claims that melatonin directly scavenges hydrogen peroxide and shown them to be unfounded. Relative hydrogen peroxide concentrations were determined in the absence and presence of melatonin using both an isoluminol-based chemiluminescence assay (with measurements at circa 40 s, 6 h, and 24 h after mixing) and the phenol red/horseradish peroxidase assay employed by two earlier groups of workers (with measurements at 5 s, then every minute for the first 5 min, and then every hour to 5 h). Both assay procedures were in agreement. There was no significant change in the hydrogen peroxide concentrations over 24 h, and, furthermore, the concentrations of H(2)O(2) in the presence and absence of melatonin were the same within experimental error. Our results were obtained in metal ion-free systems. It therefore appears likely that the claims for a direct melatonin/H(2)O(2) reaction were due to contamination by traces of transition metal ions.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.     

Chemiluminescence in the reaction of cytochrome c with hydrogen peroxide

(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H₂O₂ at pH 8.0 emit chemiluminescence with quantum yield $\text{Ф ? 10}{}^{\mathrm{?9}}$ and absolute maximum intensity $\text{I}{}_{\mathrm{<mtext>max</mtext>}}\text{? 10}{}^5\text{hv/}\text{s}$ per cm³ (λ = 440), and exhibit exponential decay with a rate constant of 0.15 s^?1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H₂O₂, a weak fluorescence in the region 400–620 nm with λ_max = 465–510 nm (λ_exc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan (λ_exc = 280 nm, λ_em = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λ_max = 530 nm (λ_exc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen $\text{O}{}_2\text{(}{}^1\text{Δ}{}_{\mathrm{<mtext>g</mtext>}}\text{)}$ is not involved in either peroxidation or chemiluminescence. (6) The haem Fe³⁺ group and H₂O₂ appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds.     

Accelerated CuZn-SOD-mediated oxidation and reduction in the presence of hydrogen peroxide

Copper, zinc-superoxide dismutase (CuZn-SOD) is a cytosolic, antioxidant enzyme that scavenges potentially damaging superoxide radical (()O(2)(-)). Under the proper conditions, CuZn-SOD also catalyzes the oxidation and reduction of certain small molecules. Here, we demonstrate that increased exposure to hydrogen peroxide (H(2)O(2)), a by-product of the ()O(2)(-) scavenging reaction, dramatically increases the ability of CuZn-SOD to oxidize melatonin and reduce S-nitrosoglutathione (GSNO). After a 15min in vitro incubation with CuZn-SOD and 1mM H(2)O(2), 76% of the melatonin was oxidized, compared to 52% with 0.25mM H(2)O(2), and just 9% without H(2)O(2). Pre-incubation with 1mM H(2)O(2) resulted in a 100% increase in the rate of GSNO breakdown by CuZn-SOD in the presence of glutathione (GSH) compared to untreated CuZn-SOD. Collectively, these data suggest that even small increases in intracellular H(2)O(2) levels may result in the oxidation and/or reduction of small molecules critical for proper cellular function.     

Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide

Horseradish peroxidase (HRP) is a commonly used enzyme in many biotechnological fields. Improvement of HRP stability would further increase its potential application range. In the present study, 13 single- and three double-mutants of solvent exposed, proximal lysine and glutamic acid residues were analysed for enhanced H(2)O(2) stability. Additionally, five single- and one pentuple-consensus mutants were investigated. Most mutants displayed little or no alteration in H(2)O(2) stability; however, three (K232N, K241F and T110V) exhibited significantly increased H(2)O(2) tolerances of 25- (T110V), 18- (K232N), and 12-fold (K241F). This improved stability may be due to an altered enzyme-H(2)O(2) catalysis pathway or to removal of potentially oxidisable residues.     

Elevated exhalation of hydrogen peroxide in patients with non-small cell lung cancer is not affected by chemotherapy

Objectives: Reactive oxygen species, which are implicated in the process of carcinogenesis, are also responsible for cell death during chemotherapy (CHT). Therefore, the aim of the study was to evaluate exhaled H₂O₂ levels in non-small cell lung cancer (NSCLC) patients before and after CHT.

Methods: Thirty patients (age 61.3?±?9.3 years) with advanced NSCLC (stage IIIB–IV) and 15 age-matched healthy cigarette smokers were enrolled into the study. Patients received four cycles of cisplatin or carboplatin with vinorelbine every three weeks. Before and after the first, second, and fourth cycle, the concentration of H₂O₂ in exhaled breath condensate was measured with respect to treatment response.

Results: At the baseline, NSCLC patients exhaled 3.8 times more H₂O₂ than the control group (0.49?±?0.14 vs. 0.13?±?0.03?µmol/L, P?2O₂ levels independent of the treatment response (partial remission vs. progressive disease). Pre- and post-CHT cycles of H₂O₂ levels generally correlated positively.

Discussion: The study demonstrated the occurrence of oxidative stress in the airways of advanced NSCLC patients. Exhaled H₂O₂ level was not affected by CHT and independent of treatment results and changes in the number of circulating neutrophils.     

Involvement of malate,monophenols, and the superoxide radical in hydrogen peroxide formation by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Peroxidase associated with isolated horseradish cell walls catalyzes the formation of H₂O₂ in the presence of NADH. The reaction is stimulated by various monophenols, especially of coniferyl alcohol. NADH can be provided by a bound malate dehydrogenase. This system is capable of polymerizing coniferyl alcohol yielding an insoluble dehydrogenation polymer. NADH was found to be oxidized by two different mechanisms, one involving Mn²⁺, monophenol, and the superoxide radical O₂ ^·- in a reaction that is not affected by superoxide dismutase, and another one depending on the presence of free O₂ ^·- and probably of an enzyme-NADH complex. A scheme of these reaction chains, which are thought to be involved in the lignification process, is presented.Abbreviations DHP dehydrogenation polymer - GOT glutamate oxaloacetate transaminase (EC 2.6.1.1) - LDH lactate dehydrogenase (pig heart, EC 1.1.1.27) - MDH malate dehydrogenase (EC 1.1.1.37) - pCA p-coumaric acid - SOD superoxide dismutase (EC 1.15.1.1) - TLC thin-layer chromatography - XOD xanthine oxidase (EC 1.2.3.2)     

Melatonin inhibits the contractile effect of vanadate in the isolated pulmonary arterial rings of rats: possible role of hydrogen peroxide

The effect and possible mechanism of action of vanadate on the isolated pulmonary arterial rings of normal rats were studied. Pulmonary arterial rings contracted in response to vanadate (0.1-1 mM) in a concentration-dependent manner. Preincubation of the pulmonary arterial rings with 1 mM melatonin significantly reduced the contractile effect of vanadate by more than 60%. Furthermore, addition of hydrogen peroxide (50 microM) or enzymatic generation of hydrogen peroxide by the addition of glucose oxidase (10 U/mL) to the medium containing glucose produced remarkable increases in the pulmonary arterial tension, 46.2 +/- 7.3 and 78.7 +/- 9.7 g tension/g tissue, respectively. Similarly, incubation of the pulmonary arterial rings with 1 mM melatonin significantly reduced the contractile responses of the arterial rings to hydrogen peroxide and glucose/glucose oxidase to 25.7 +/- 2.9 and 24.7 +/- 4.4 g tension/g tissue, respectively. Vanadate, in vitro, significantly stimulated the oxidation of NADH by xanthine oxidase, and the rate of oxidation was increased by increasing either time or vanadate concentration. Similarly, addition of melatonin to a reaction mixture containing xanthine oxidase and vanadate significantly inhibited the rate of NADH oxidation in a concentration-dependent fashion. The results of the present study indicated that vanadate induced contraction in the isolated pulmonary arterial rings, which was significantly reduced by melatonin. Furthermore, the contractile effect of vanadate on the pulmonary arterial rings may be attributed to the intracellular generation of hydrogen peroxide.     

Melatonin does not produce sedation in rats: A chronobiological study

ABSTRACT

Background: Melatonin has been associated with a wide variety of cellular, neuroendocrine, and neurophysiological processes. Clinical studies have reported the use of melatonin as an agent that exerts sedative-hypnotic effects. However, evidence of the sedative-hypnotic effects of different doses of melatonin is inconsistent, and available data regarding its night/day-time sedative effects are limited. The purpose of this study was to evaluate the effects of melatonin administered at different times of day on the magnitude of the sedative-hypnotic activity of different melatonin doses (5, 10, 30, and 50 mg/kg) in rats.

Methods: Sedation was assessed in Wistar rats behaviorally, using rota-rod, spontaneous locomotor activity, and fixed-bar tests at different times of day (ZT4, ZT10, ZT16, and ZT22).

Results: Our results showed that, compared to trazodone, acute and chronic dosing of ≤5 mg melatonin produced mild, transient sedative effects, mainly in the light period. Nevertheless, doses of ≥10 mg/kg did not cause sustained sedative effects.

Conclusion: These results suggest that melatonin may be used for sedation induction, mainly in preoperative patients.     

Effect of exogenous hydrogen peroxide on enzymatic and nonenzymatic antioxidants in leaves of young pea plants treated with paraquat

The effects of exogenously applied hydrogen peroxide on the antioxidant system of pea plants were investigated. Ten-day-old pea seedlings were sprayed with 2.5 mM H₂O₂ and 24 h later with 0.2 mM PQ. Samples were taken 0, 2 and 5 h after the start of illumination. The protective effect of H₂O₂ was evaluated by monitoring of parameters related to the damage caused by PQ. The treatment with PQ led to a severe leakage of electrolytes from leaf tissues. Malondialdehyde level increased in PQ treated plants, but remained unchanged in H₂O₂ pre-treated ones after 5 h of illumination. Increased catalase and glutathione-S-transferase activity was observed in pea plants treated with H₂O₂ and PQ. Ascorbate peroxidase activity decreased significantly after paraquat application, but pre-treatment with H₂O₂ prevented ascorbate peroxidase inhibition to some extent. Increased guaiacol peroxidase activity was detected after H₂O₂ application. PQ application caused a drastic decline in the levels of thiol-group bearing compounds, reduced glutathione and ascorbate, while the quantity of oxidized glutathione and dehydroascorbate were increased. The results presented on changes in enzymatic and nonenzymatic antioxidants suggest that preliminary H₂O₂ application to pea plants treated with PQ, alleviates the toxic effects of the herbicide.