The formation of hydrogen peroxide during the oxidation of reduced nicotinamide adenine dinucleotide by cytochrome o from Vitreoscilla.

The formation of hydrogen peroxide during the oxidation of NADH by purified preparations of cytochrome o has been demonstrated by employing three independent methods: polarographic, colorimetric, and fluorometric. The first two methods were used to assay for the accumulation of hydrogen peroxide and showed that hydrogen peroxide did accumulate as a product, but only about 30% of the oxygen consumed or 15 to 20% of the NADH oxidized was recoverable as hydrogen peroxide. This lack of 1:1 stoichiometry was not due to residual catalase activity in these preparations which could be eliminated by freeze-thawing. Thus, hydrogen peroxide may not be the sole or primary product of the NADH-cytochrome o oxidase reaction. The fluorometric assay could be coupled directly to the NADH-cytochrome o oxidase reaction in one medium, and this method showed that hydrogen peroxide was generated continuously from the beginning of the reaction in a 1:1 stoichiometry, hydrogen peroxide generated to NADH oxidized. This result suggests that hydrogen peroxide is an intermediate that can be trapped efficiently under the conditions of the fluorometric assay, whereas under the conditions of the first two assays most of the hydrogen peroxide generated undergoes further reaction. Exogenously added FAD or FMN increased the percentage of hydrogen peroxide that accumulated in the NADHcytochrome o oxidase reaction. Flavin is believed to act on the reductase side of cytochrome o so the increased percentage of hydrogen peroxide is not likely to result from the direct reaction of reduced flavin with oxygen.     

Corn fiber hydrolysis by<Emphasis Type="Italic"> Thermobifida fusca</Emphasis> extracellular enzymes

Thermobifida fusca was grown on cellulose (Solka-Floc), xylan or corn fiber and the supernatant extracellular enzymes were concentrated. SDS gels showed markedly different protein patterns for the three different carbon sources. Activity assays on a variety of synthetic and natural substrates showed major differences in the concentrated extracellular enzyme activities. These crude enzyme preparations were used to hydrolyze corn fiber, a low-value biomass byproduct of the wet milling of corn. Approximately 180 mg of reducing sugar were produced per gram of untreated corn fiber. When corn fiber was pretreated with alkaline hydrogen peroxide, up to 429 mg of reducing sugars were released per gram of corn fiber. Saccharification was enhanced by the addition of beta-glucosidase or by the addition of a crude xylanase preparation from Aureobasidium sp.     

Glutamine synthetase from the green alga Monoraphidium braunii is regulated by oxidative modification

Monoraphidium braunii glutamine synthetase is inactivated by several mixed-function oxidation systems. Inactivation requires oxygen and a metal cation as it does not take place under anaerobic conditions or in the presence of EDTA. Glutamine synthetase can be protected against that inactivation by peroxidase and catalase but not by superoxide dismutase indicating that hydrogen peroxide is involved in the process, although hydrogen peroxide is not itself effective. The oxidative modification of glutamine synthetase renders the protein more sensitive to temperature and susceptible to proteolytic attack. This has been demonstrated by measuring by quantitative immunoelectrophoresis the levels of glutamine synthetase antigen, in enzymatic preparations treated with different oxidation systems. Besides, immunoblotting of crude extracts in the presence of mixed-function oxidation systems shows the disappearance of material cross-reacting with anti-glutamine synthetase antibodies. Other results show that glutamine synthetase from Chlamydomonas reinhardtii could be subjected to the same kind of oxidative inactivation. The possible regulatory role of oxidative modification of glutamine synthetase in green algae is discussed.     

Colorimetric alcohol assays with alcohol oxidase

A method is described for manufacturing crude alcohol oxidase (EC 1.1.3.13) preparations which are suitable for application in colorimetric alcohol assays. The procedure involves a one-step removal of catalase activity from a partially purified preparation of alcohol oxidase from the yeast Hansenula polymorpha via dialysis against 3-amino-1,2,4-triazole and hydrogen peroxide. Thus, the irreversible inactivation of more than 90% of the catalase present was achieved, which is prerequisite for the use of alcohol oxidase preparations in colorimetric alcohol assays via peroxidase-mediated oxidation of dyes. This type of assay was shown to be rapid, accurate and sensitive. The influence of the relative concentrations of the various assay constituents such as alcohol oxidase, catalase and peroxidase is discussed. It is concluded that this colorimetric alcohol assay is particularly suitable for the determination of ethanol in fermentation broths, both in qualitative and in quantitative tests.     

Enzymatic transformations of morphinane alkaloids

Horseradish peroxidase transforms morphinane alkaloids into N-oxides and morphine to pseudomorphine in the presence of hydrogen peroxide. The crude poppy enzyme fraction shows the same activities. The rates of reactions were influenced by phenolic compounds and their relation controlled by the concentration of hydrogen peroxide and the presence of ascorbic acid.     

A relation between G-, C-, and N-band patterns as revealed by progressive oxidation of chromosomes and a note on the nature of N-bands

Near-ultraviolet irradiation of chromosome preparations mounted in a hydrogen peroxide solution resulted in an oxidative disintegration of the structure of fixed metaphase chromosomes with concomitant production of various band patterns appearing after staining with Giemsa. Neither irradiation nor hydrogen peroxide alone could produce banding. After irradiation in the presence of hydrogen peroxide the gradually increasing effect of oxidation on the chromosomes along the gradient of light intensities from the periphery of the slide towards the radiation focus in the centre of the slide became visible as G-, C-, and N-banding, respectively. Close to the centre only contours of chromosomes were left after this treatment. Although G-banding and differential DNA-extraction often went together, extraction of DNA was not an absolute requirement to obtain a G-band pattern. N-bands appeared to be the chromosomal regions that were most resistant to destruction. Staining methods specific for DNA failed to demonstrate these bands, although with Giemsa an intense staining reaction occurred. On the analogy of the staining behaviour of model protein preparations with Giemsa a phosphoprotein nature is suggested for the N-band material in the chromosomes.     

Oxidation of p-coumaroylagmatine in barley seedling extracts in the presence of hydrogen peroxide or thiols

When p-coumaroylagmatine is oxidized in the presence of hydrogen peroxide in a crude extract of barley seedlings, among several products, hordatine A is formed. However, unlike the natural isomer, this is optically inactive. In the absence of hydrogen peroxide and when a thiol (glutathione, cysteine, mercaptoethanol or dithiothreitol) is added to the incubation medium, p-coumaroylagmatine is rapidly transformed to a thiol adduct, probably through a peroxidase-dependent co-oxidation reaction. The reactions with hydrogen peroxide or with a thiol are completely inhibited by 1 mM ascorbate.     

A more sensitive modification of the catalase assay with the Clark oxygen electrode : Application to the kinetic study of the pea leaf enzyme

A modification of the method of catalase determination by means of the Clark oxygen electrode is described. The assay is based on measurement of the initial rate at which oxygen is released by catalase in an oxygen-free buffer. Displacement of oxygen was brought about by flushing with nitrogen, and the substrate used was hydrogen peroxide at a 33.5 m final concentration. The method is rapid and can be used with crude catalase preparations. Its sensitivity is at least 20 times higher than that of previous methods; it has an interval of measurable activity of about 0.01–8.4 μmol of O₂/min and, therefore, is applicable to an 840-fold range of catalase concentrations. This modification was applied to the kinetic study of crude extracts of pea leaf catalase. An apparent K_m of 0.190 was calculated.     

A more sensitive modification of the catalase assay with the Clark oxygen electrode: Application to the kinetic study of the pea leaf enzyme

A modification of the method of catalase determination by means of the Clark oxygen electrode is described. The assay is based on measurement of the initial rate at which oxygen is released by catalase in an oxygen-free buffer. Displacement of oxygen was brought about by flushing with nitrogen, and the substrate used was hydrogen peroxide at a 33.5 mm final concentration. The method is rapid and can be used with crude catalase preparations. Its sensitivity is at least 20 times higher than that of previous methods; it has an interval of measurable activity of about 0.01–8.4 μmol of O₂/min and, therefore, is applicable to an 840-fold range of catalase concentrations. This modification was applied to the kinetic study of crude extracts of pea leaf catalase. An apparent K_m of 0.190 m was calculated.     

Effect of thymic peptides on the functional activity of phagocytic cells of donor peripheral blood

The direct action of synthetic peptide preparations, analogous to thymic hormones, on the functions of phagocytic cells was studied. The preparations Thymogen, Neogen and Thymodepressin in a dose of 10 mM produced a stimulating effect on the ingestive activity of the neutrophil, but not monocytic, population. All three preparations also enhanced the formation of oxygen metabolites registered in the luminol-dependent chemiluminescent analysis. The characteristics of spontaneous chemiluminescence (CL) reflecting the basal level of the synthesis of the active forms of oxygen and CL induced by opsomized zymosan significantly increased also in those cases when the preparations were used in a dose of 10 mM. The level of the synthesis of hydrogen peroxide in individual cells could be appraised by the intensity of the luminescence of dichlorofluorescein diacetate (DCF-DA), evaluated with the use of flow cytometry. All preparations produced a stimulating effect on the formation of hydrogen peroxide in monocytes. The reaction of neutrophils was even more active: Neogen (10 mM) produced the twofold change in the intensity of the luminescence of DCF-DA) in neutrophils, Thymogen and Thynodepressin increased the average intensity of the luminescence of DCF-DA by 80% and 60%, respectively.     

Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species

Extracellular peroxidases are classified as free, or ionically or covalently bound to the cell wall. In addition, peroxidase-like activities have often been demonstrated at the outer surface of protoplasts and plasma membrane preparations. Under certain conditions apoplastic peroxidases have been shown to contribute to the formation of superoxide and hydrogen peroxide during the `oxidative burst' through the oxidation of a reductant. However, the identity of this reductant remains unclear. It has been suggested that the production of these active oxygen species may play important roles in plant responses to biotic and abiotic stress. Extracellular release of pre-existing and de novo synthesis of apoplastic peroxidases is regulated by changing environmental conditions. While the oxidative burst could potentially be harmful to a plant's own cells, tissues can rapidly metabolize even high concentrations of hydrogen peroxide. Recent work has shown that when extracellular hydrogen peroxide exceeds the supplies of reductants, class II and class III peroxidases can display catalase-like activity. Under these conditions, hydrogen peroxide is able to act as both oxidizing and reducing substrate. It seems likely therefore, that a further role of extracellular peroxidases is to protect plants from the consequences of the oxidative burst that they themselves are responsible for producing.     

Oxidation of Alcohols by Botrytis cinerea

Crude cell-free preparations of Botrytis cinerea were found to oxidize straight-chain primary alcohols (except methanol), aromatic primary alcohols, and unsaturated primary alcohols. The resulting products were the corresponding aldehydes and an equal molar quantity of hydrogen peroxide.     

Phenol removal from aqueous solutions by peroxidase extracted from horseradish

Horseradish peroxidase (HRP) is one of the most recently used enzymes in the process of enzymatic phenol removal. It has a catalytic ability over a broad range of pH, temperature and contaminant concentrations. In this study we revealed the possibility of successful use the crude peroxidase obtained from horseradish roots for the phenol removal from aqueous solutions in the presence of the low molecular polyethylene glycol (PEG 300) at room temperature (20°C) and pH 7.2. Reaction was monitored by direct measuring of the absorbance changes in a samples taken at certain time intervals from the reaction mixture. At the first time PEG 300 was shown to be a more stabilizing effect on crude HRP and provided a higher phenol removal in comparison with PEG 3350. Crude HRP used in these study demonstrated a greater resistance on phenol and hydrogen peroxide inactivation that allowed a higher phenol removal. The highest phenol removal was achieved when the concentration of PEG 300, phenol and hydrogen peroxide were 300 mg/L, 2.0 and 2.5 mM, respectively.     

Role of oxygen and metal ions in the instability of streptolysin O.

Thiol-containing preparations of streptolysin O (SLO) and pure cysteine generate superoxide radicals in alkaline buffer on autoxidation of the thiol groups. Autoxidation is stimulated by cupric ions. Reconstituted SLO preparations accumulate hydrogen peroxide with a concomitant loss of activity on storage at room temperature. Short-term protection of hemolytic activity was achieved by inclusion of catalase in the preparation; no apparent protection was observed by superoxide dismutase, whereas 1,10-O-phenanthroline offered long-term protection of the hemolysin.     

Effect of destruction of Escherichia coli cells on the catalytic ability of catalase

The possibility for investigation of catalase (CAT) activity under the conditions of intact E. coli cells was estimated. This approach is based on the possibility of hydrogen peroxide freely cross biological membranes. CAT activity of native cells had a broad maximum between pH values 4.5 and 7.5. Desintegration of cells by freezing--thawing and ultrasonication indicated that there were two CAT activity peaks at pH values about 3.5 and 7.0. Activity of CAT with acid pH-optimum decreased at cell desintegration, but one with neutral pH-optimum was rather stable under this procedure. The enzyme in native conditions was less sensitive to the inhibition by high concentrations of hydrogen peroxide than its counterpart from destroyed cells. Activity of CAT in native and desintegrated cell preparations had different sensitivity to heating and inhibition by reduced glutathione, but it was inhibited by azide similarly. Difference in the CAT properties of native and desintegrated bacteria preparations may be explained by different possibility to penetrate cell membrane by reagents and/or by possible modification of the enzyme properties at destruction of native microenvironment.     

From detergent additive to semisynthetic peroxidase-simplified and up-scaled synthesis of seleno-subtilisin

A simplified and up-scaled synthesis of the semisynthetic peroxidase seleno-subtilisin was developed. Highly purified to technical grade subtilisin preparations from Bacillus licheniformis and Bacillus amyloliquefaciens were applied as starting materials. Activation of Ser 221 with phenylmethanesulfonyl fluoride, nucleophilic substitution by sodium hydrogen selenide, and oxidation to the seleninic acid with hydrogen peroxide completed the chemical active-site modification. The reactions were accomplished with a minimum of simple work-up procedures in 10 g scale. Kinetics and enantioselectivity of the preparations were tested using 1-phenylethyl hydroperoxide. For the first time, an up-scaled synthesis of a semisynthetic enzyme is available. Copyright 1998 John Wiley & Sons, Inc.     

Haemonchus contortus utilises catalase in defence against exogenous hydrogen peroxide in vitro

The toxicity of activated oxygen species towards adult Haemonchus contortus nematodes was examined in in vitro assays using ingestion of [³H]inulin to assess nematode viability. Both glucose/glucose oxidase (generation of hydrogen peroxide) and xanthine/xanthine oxidase (generation of superoxide anion) systems showed concentration-dependant toxicity to the nematodes. Both adult and larval Haemonchus contortus enzyme preparations showed significant catalase activities. Adult nematodes exposed to aminotriazole for 24 h showed catalase activities reduced to less than 20% of controls. Aminotriazole-treated nematodes exposed to a glucose/glucose oxidase system were significantly more susceptible to the toxic effects of the oxidant-generating system than controls (no aminotriazole pre-treatment). The concentration of glucose oxidase required to inhibit feeding by 50% was decreased 33-fold in aminotriazole-treated nematodes compared with controls. The effect of aminotriazole pre-treatment implicates hydrogen peroxide as a significant toxic agent in the glucose/glucose oxidase system. It is apparent that inhibition of Haemonchus contortus catalase increases the susceptibility of the parasite to the toxic effects of hydrogen peroxide, demonstrating a protective role for this enzyme. This suggests that catalase has the potential to play a significant role in the defence of this parasite against hydrogen peroxide produced as part of the respiratory burst of activated phagocytes within the host during its response to nematode infection.     

Biosynthesis of D-glucaric acid in mammals: a free-radical mechanism?

In the presence of iron salts and hydrogen peroxide, D-glucuronic acid was converted into D-glucaric acid. The reaction was strongly inhibited by free-radical scavengers and is ascribed to the action of the hydroxyl radical. The formation of D-glucarate was dependent upon pH and occurred in the presence of some iron-complexing agents. The first product of oxidation was a lactone that was a strong inhibitor of beta-D-glucuronidase and assumed to be D-glucaro-1,5-lactone. Microsomal preparations in the presence of NADPH also produced D-glucarate from D-glucuronic acid, presumably due to formation of hydrogen peroxide, and the product was an inhibitor of beta-D-glucuronidase. Superoxide did not produce D-glucarate from D-glucuronate. The cytochrome P450 system is more likely than "glucuronolactone dehydrogenase" to be responsible for the production of D-glucaric acid in vivo.     

Comparative sporicidal effects of liquid chemical agents.

We compared the effectiveness of glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate (plus a sublethal amount of hydrogen peroxide), sodium hypochlorite, and phenol to inactivate Bacillus subtilis spores under various conditions. Each chemical agent was distinctly affected by pH, storage time after activation, dilution, and temperature. Only three of the preparations (hypochlorite, peracetic acid, and cupric ascorbate) studied here inactivated more than 99.9% of the spore load after a 30-min incubation at 20 degrees C at concentrations generally used to decontaminate medical devices. Under similar conditions, glutaraldehyde inactivated approximately 90%, and hydrogen peroxide, formaldehyde, and phenol produced little killing of spores in suspension. By kinetic analysis at different temperatures, we calculated the rate of spore inactivation (k) and the activation energy of spore killing (delta E) for each chemical agent. Rates of spore inactivation had a similar delta E value of approximately 20 kcal/mol (ca.83.68 kJ/mol) for every substance tested. The variation among k values allowed a quantitative comparison of liquid germicidal agents.     

Direct electrochemistry and electrocatalytic activity of catalase immobilized onto electrodeposited nano-scale islands of nickel oxide

Cyclic voltammetry was used for simultaneous formation and immobilization of nickel oxide nano-scale islands and catalase on glassy carbon electrode. Electrodeposited nickel oxide may be a promising material for enzyme immobilization owing to its high biocompatibility and large surface. The catalase films assembled on nickel oxide exhibited a pair of well defined, stable and nearly reversible CV peaks at about -0.05 V vs. SCE at pH 7, characteristic of the heme Fe (III)/Fe (II) redox couple. The formal potential of catalase in nickel oxide film were linearly varied in the range 1-12 with slope of 58.426 mV/pH, indicating that the electron transfer is accompanied by single proton transportation. The electron transfer between catalase and electrode surface, (k(s)) of 3.7(+/-0.1) s(-1) was greatly facilitated in the microenvironment of nickel oxide film. The electrocatalytic reduction of hydrogen peroxide at glassy carbon electrode modified with nickel oxide nano-scale islands and catalase enzyme has been studied. The embedded catalase in NiO nanoparticles showed excellent electrocatalytic activity toward hydrogen peroxide reduction. Also the modified rotating disk electrode shows good analytical performance for amperometric determination of hydrogen peroxide. The resultant catalase/nickel oxide modified glassy carbon electrodes exhibited fast amperometric response (within 2 s) to hydrogen peroxide reduction (with a linear range from 1 microM to 1 mM), excellent stability, long term life and good reproducibility. The apparent Michaelis-Menten constant is calculated to be 0.96(+/-0.05)mM, which shows a large catalytic activity of catalase in the nickel oxide film toward hydrogen peroxide. The excellent electrochemical reversibility of redox couple, high stability, technical simplicity, lake of need for mediators and short preparations times are advantages of this electrode. Finally the activity of biosensor for nitrite reduction was also investigated.