Polyaniline based catalase biosensor for the detection of hydrogen peroxide and azide

The CAT/PANi/ITO bioelectrode has been prepared as a catalase biosensor and shows response for monitoring not only of H₂O₂ but also azide. The sensor exhibited an excellent response to the H₂O₂ and azide. The linear range of H₂O₂ was 0.064∼1 mM and for azide 0.125∼4 mM, respectively. Catalase biosensor was based on the principle of the measurements as the decrease in the differentiation of oxygen level, which has been caused by the inhibition of catalase in the bioactive layer of the biosensor by azide. The repeatability experiments were done in triplicate. The logarithm response of the biosensor to H₂O₂ (r² = 0.99), as well as, for azide (r² = 0.90) were reported, respectively. The bioelectrode was characterized by CV and AFM. The proposed biosensor would be applied for the determination of H₂O₂ and azide in various biological samples.     

Mechanism of cytochrome c peroxidase. O-benzoylhydroxylamine as an analog of hydrogen peroxide.

A number of reagents, some of which are electronic analogs of hydrogen peroxide, will replace it in the reactions of cytochrome c peroxidase. These compounds include N-bromosuccinimide, sodium hypochlorite, and the novel oxidizing agent O-benzoylhydroxylamine. If fragments of the oxidant played a functional role in the structure of the oxidized form of the enzyme, it would be expected that the product formed from O-benzoylhydroxylamine would differ from that formed from hydrogen peroxide. The products formed on reaction of the two oxidizing agents with cytochrome c peroxidase are indistinguishable. This results carries implications for the structure of the so-called ES compound. The extension in the range of specific substrates for cytochrome c peroxidase allows identification of the structure which compounds must possess to be oxidizing substrates for the enzyme. A mechanism for the first step of the reaction is suggested. O-Benzoylhydroxylamine is also a reducing agent, and its reaction with the enzyme is analogous to that of hydrogen peroxide with catalase. The final product of the reaction is the inert nitric oxide complex of ferrous cytochrome c peroxidase.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

New fluorogenic substrates for horseradish peroxidase: Rapid and sensitive assays for hydrogen peroxide and the peroxidase

p-Hydroxyphenyl compounds [3-(p-hydroxyphenyl)propionic acid, p-hydroxyphenethyl alcohol, hordenine, p-ethylphenol, 3-(p-hydroxyphenyl)-1-propanol, p-n-propylphenol, and p-hydroxyphenyllactic acid] were recently found to be excellent fluorogenic substrates for the horseradish peroxidase-mediated reaction with hydrogen peroxide. A very rapid and sensitive method for the fluorometric assays of hydrogen peroxide and the peroxidase was established by using 3-(p-hydroxyphenyl)propionic acid as the best of these substrates; hydrogen peroxide can be assayed precisely in amounts as small as 0.1 nmol, with peroxidase activity as low as 7.8 μU.     

Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide

It is well known that catalase is transformed to nitric oxide-Fe2+-catalase by hydrogen peroxide (H2O2) plus azide. In this report, we show that myeloperoxidase is also inactivated by H2O2 plus azide. Utilizing this system, we studied the presence and source of intracellular H2O2 generated by activated neutrophils. Stimulation of neutrophils with phorbol myristate acetate (PMA, 100 ng/ml) plus azide (5 mM) for 30 min completely inactivated intragranular myeloperoxidase and reduced cytosolic catalase to 35% of resting cells. This intracellular inactivation of heme enzymes did not occur in normal neutrophils incubated with either PMA or azide alone or in neutrophils from patients with chronic granulomatous disease (CDG) which cannot produce H2O2 in response to PMA. Incubation of neutrophils with azide and a H2O2 generating system (glucose-glucose oxidase) inactivated 41% of neutrophil myeloperoxidase. Glutathione-glutathione peroxidase (GSH-GSH peroxidase), an extracellular H2O2 scavenger, totally protected neutrophil myeloperoxidase from inactivation by azide plus glucose-glucose oxidase. In addition, when a mixture of normal and CGD cells was stimulated with PMA in the presence of azide, 90% of the myeloperoxidase in CGD neutrophils was inactivated. Therefore, H2O2 released extracellularly from activated neutrophils can diffuse into cells. In contrast, myeloperoxidase in normal polymorphonuclear leukocytes stimulated with PMA in the presence of azide and GSH-GSH peroxidase was 75% inactivated. Thus, the results indicate that a GSH-GSH peroxidase-insensitive pool of H2O2 is also generated, presumably at the plasma membrane, and this pool of H2O2 can undergo direct internal diffusion to inactivate myeloperoxidase.     

Use of hydrogen peroxide for closing disulfide bridges in peptides

The use of hydrogen peroxide for the formation of disulfide bridges was studied in 15 peptides of various lengths and structures. The oxidation of peptide thiols by hydrogen peroxide was shown to proceed under mild conditions without noticeable side reactions of Trp, Tyr, and Met residues. Yields of the corresponding cyclic disulfides were high and mostly exceeded those obtained with other oxidative agents, in particular, iodine. It was established that the use of hydrogen peroxide in organic medium also provided sufficiently high yields when large-scale syntheses of oxytocin and octreotide (up to 10 g) were carried out. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Lipid membrane immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

Stable films of didodecyldimethylammonium bromide (DDAB, a synthetic lipid) and horseradish peroxidase (HRP) were made by casting the mixture of the aqueous vesicle of DDAB and HRP onto the glassy carbon (GC) electrode. The direct electron transfer between electrode and HRP immobilized in lipid film has been demonstrated. The lipid films were used to supply a biological environment resembling biomembrane on the surface of the electrode. A pair of redox peaks attributed to the direct redox reaction of HRP were observed in the phosphate buffer solution (pH 5.5). The cathodic peak current increased dramatically while anodic peak decreased by addition of small amount H(2)O(2). The pH effect on amperometric response to H(2)O(2) was studied. The biosensor also exhibited fast response (5 s), good stability and reproducibility.     

An improved method for the inhibition of endogenous peroxidase non-deleterious to lymphocyte surface markers. Application to immunoperoxidase studies on eosinophil-rich tissue preparations

Summary The inhibitory effect of phenylhydrazine and azide combined with either pre-formed or nascent hydrogen peroxide H₂O₂ upon endogenous peroxidatic activity, expressed by tissue eosinophils in different disease states, was investigated. It was found that whilst endogenous peroxidatic activity due to eosinophils in a Hodgkin's disease and a histiocytosis X case were adequately inhibited by phenylhydrazine combined with pre-formed or nascent H₂O₂, the eosinophils in theOnchocerca volvulus nodule were either not at all or only partly inhibited by the two regimens. On the other hand, a combination of azide with nascent H₂O₂ proved consistently effective against this resistant form of endogenous peroxidatic activity. Using human tonsil sections this protocol was shown to be non-deleterious to T₄(CD4), T₆(CD1) and T₈(CD8) lymphocyte surface antigens as evidenced by the application of a standard indirect immunoperoxidase technique and the relevant monoclonal antibodies.     

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.     

The mechanism of oxyperoxidase formation from ferryl peroxidase and hydrogen peroxide

Formation of oxyperoxidase from the reaction of ferryl horseradish peroxidase with H2O2 is inhibited by a small amount of tetranitromethane (TNM), a powerful scavenger of superoxide anion radical. The inhibition by TNM, however, does not exceed 35% as the TNM concentration is increased above 5 microM. The stoichiometry of the reaction in the presence of TNM suggests the following equation for TNM-sensitive formation of oxyperoxidase. Ferryl peroxidase + H2O2----(ferric peroxidase + O2- + H+)----oxyperoxidase The kinetic study on the TNM-resistant formation of oxyperoxidase suggests that the displacement of the oxygen with H2O2 takes place at the sixth coordination position at maximal rates of 0.048 and 0.054 s-1 for peroxidases A and C, respectively, at 5 degrees C. The TNM-sensitive and -resistant reactions are concluded to occur in parallel, and both yield oxyperoxidase. In either mechanism, the protonated form of ferryl peroxidase is active and the pK alpha value is 7.1 for peroxidase A and 8.6 for peroxidase C. Oxyperoxidase decomposes spontaneously with a large activation energy (23.0 kcal/mol), and the reaction of ferryl peroxidase with H2O2 reaches a steady level of oxyperoxidase, which depends on pH and the concentration of H2O2.     

Salicylic acid,hydrogen peroxide and calcium-induced saline tolerance associated with endogenous hydrogen peroxide homeostasis in naked oat seedlings

This study investigates the role of salicylic acid (SA), hydrogen peroxide (H₂O₂) and calcium chloride (CaCl₂) singly or in combination, in inducing naked oat plant tolerance to sodium chloride (NaCl). Two-week-old naked oat plants were pretreated with both single and double of 0.5 mM SA, 0.5 mM H₂O₂ and 5 mM CaCl₂ by adding them to the culture solution for 24 h. At the end of the pretreatment, the plants were subjected to 200 mM NaCl exposure for 7 days. Data were collected on plant biomass, H₂O₂ level, antioxidant enzyme activity, non-enzymatic antioxidant content and malondialdehyde (MDA) content. Results showed that exposure to salt significantly inhibited plant growth, and the shoot and root dry weights were reduced 47.5% and 63.4%, and the H₂O₂ levels elevated 5.8 and 2.4 times in comparison with those in the control, respectively. Under the saline stress, the activities of superoxide dismutase (SOD) and catalase (CAT) were induced, but the contents of ascorbic acid (AA) and glutathione (GSH) decreased, and MDA largely accumulated. The various pretreatments efficiently counteracted the salt-caused growth inhibition, especially with H₂O₂ + CaCl₂ the shoot and root dry weights reduced only 9.4% and 24.4% of the non salt-stressed plants. The determination of endogenous H₂O₂ level demonstrated that the pretreatments induced H₂O₂ accumulation, with H₂O₂ + CaCl₂ being most efficient, but the effect was transient. After 7 days of saline stress, the H₂O₂ contents in the pretreated shoots and roots accounted for 23.7–41.8% and 31.7–57.3% of the non-pretreated plants, varying according to the different pretreatments. Under saline stress, SOD and CAT further increased, AA and GSH maintained higher levels and MDA decreased in the pretreated plants compared to the untreated plants. With application of diphenylene iodonium (DPI) during the pretreatment, which inhibited the accumulation of H₂O₂, the ameliorative effect of the pretreatment on salt-caused plant growth inhibition was reduced. However, applied DPI at the immediate end of the pretreatment did not alter its favorable role, indicating a H₂O₂ peak formed at the early time of saline stress might play an important role in regulating plant tolerance to saline stress.     

Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide

The reaction of Euphorbia characias latex peroxidase (ELP) with hydrogen peroxide as the sole substrate was studied by conventional and stopped-flow spectrophotometry. The reaction mechanism occurs via three distinct pathways. In the first (pathway I), ELP shows catalase-like activity: H2O2 oxidizes the native enzyme to compound I and subsequently acts as a reducing substrate, again converting compound I to the resting ferric enzyme. In the presence of an excess of hydrogen peroxide, compound I is still formed and further reacts in two other pathways. In pathway II, compound I initiates a series of cyclic reactions leading to the formation of compound II and compound III, and then returns to the native resting state. In pathway III, the enzyme is inactivated and compound I is converted into a bleached inactive species; this reaction proceeds faster in samples illuminated with bright white light, demonstrating that at least one of the intermediates is photosensitive. Calcium ions decrease the rate of pathway I and accelerate the rate of pathways II and III. Moreover, in the presence of calcium the inactive stable verdohemochrome P670 species accumulates. Thus, Ca2+ ions seem to be the key for all catalytic pathways of Euphorbia peroxidase.