Presence Of Peroxidase Research Articles

Enzymatic epoxidation and polymerization of cardanol obtained from a renewable resource and curing of epoxide-containing polycardanol

Epoxide-containing polycardanol was enzymatically synthesized via two routes using two different enzymes, viz. lipase and peroxidase. Lipase catalysis was used for the epoxidation of the unsaturated alkyl chains of both cardanol and polycardanol. Peroxidase catalysis was used for the polymerization of both cardanol and epoxide-containing cardanol. One route was the synthesis of epoxide-containing cardanol from cardanol, hydrogen peroxide and an organic acid in the presence of lipase, followed by the polymerization of the phenolic functional groups of cardanol using peroxidase. In the other route, polymerized cardanol was prepared from cardanol and hydrogen peroxide in the presence of peroxidase and, subsequently, the epoxide-containing polycardanol was synthesized from polycardanol, hydrogen peroxide and an organic acid in the presence of lipase. NMR and IR spectroscopy confirmed the polymer structure, and the former route yielded epoxide-containing polycardanol in a higher yield of over 90%. The curing of the resulting polymers proceeded thermally at 150 °C, yielding transparent polymeric films with a high gloss surface within 3 h. The pencil scratch hardness of the present films was improved compared with that of polycardanol. Owing to the epoxide contained in the polymerized cardanol, the film cured with phenalkamine showed a higher hardness value after a relatively short curing time.

A simple colorimetric method for determination of hydrogen peroxide in plant tissues

A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 µmol g−1 FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method.

Polymerisation of added coniferyl alcohol by inherent xylem peroxidases and its effect on fungal decay resistance of Norway spruce

It is known that active peroxidase isozymes exist in mature wood of Norway spruce (Picea abies L. Karst.) and that they remain active for years and are found even in the heartwood (in Scots pine), where all cellular activity has ceased. This peroxidase activity was utilised in the impregnation of wood blocks with a natural monolignol, coniferyl alcohol and hydrogen peroxide. The hypothesis was that the internal wood peroxidases would oxidise the added monolignol and bind it stably into the cell wall matrix, which could hinder fungal decay. Since coniferyl alcohol is not very soluble in water, the impregnation was done under vacuum with an acetone–water solution containing 10% coniferyl alcohol and 0.4 mM H2O2 at room temperature (ca. 0.02 g of coniferyl alcohol was added to 1 g of wood). After impregnation, dimers of coniferyl alcohol and free coniferyl alcohol were found in acetone extracts with GC–MS analysis. Penetration of coniferyl alcohol and non-extractable reaction products were studied from the wood blocks with FTIR PAS technique. The wood samples treated were also subjected to a fungal decay test with Coriolus versicolor. This treatment hindered fungal decay in a 60-day experiment and led to a dry weight loss of 8.8% in comparison with 19.9% in the control. The reactions of coniferyl alcohol and H2O2 in the presence of peroxidases are discussed as well as the use of monolignols to increase wood decay resistance.

The role of benzoate secreted by Desulfotomaculum acetoxidans DSM 771 in sulfate uptake.

This work was designed to find the cause of the delay in hydrogen sulfide dissimilation in Desulfotomaculum acetoxidans DSM 771, which is dependent on the sulfate uptake. This bacterium grown without addition of any aromatic compound was shown by spectrum analysis with the methylene method to contain hydroxy-benzoate derivatives. The presence of these compounds was confirmed by HPLC in fractions obtained from cell walls after 15 days of culture. The test with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt seemed to indicate the presence of peroxidase, which probably oxidized benzoate to its hydroxy derivatives. The test with 5-sulfo-salicylic acid proved the ability of the investigated strain to utilize arylsulfates and to reduce sulfate group to hydrogen sulfide. On the basis of the above data, we propose the following sequence of reactions: 1, benzoate secretion; 2, benzoate hydroxylation; 3, sulfonation of hydroxy-benzoate derivatives.

Antioxidants Increase Apoptosis in Eutopic Endometrium of Subjects With Endometriosis

Antioxidants Increase Apoptosis in Eutopic Endometrium of Subjects With Endometriosis

Enzymatic-spectrophotometric determination of sucrose in coffee beans

A spectrophotometric method for determining sucrose is proposed. Sucrose is hydrolyzed by invertase into glucose and fructose. Then, glucose is oxidized in presence of glucose oxidase and the produced hydrogen peroxide reacts with phenol-4-sulfonic acid sodium salt and 4-aminoantipyrine in presence of peroxidase, yielding a pink dye with an absorption maximum at 505 nm. This method was validated following the EURACHEM and VAM project guidelines for method validation. Trueness, precision, robustness, sensitivity and linearity were considered. The method was applied to the determination of sucrose in green and roasted coffee beans. A comparison with the HPLC method with pulsed amperometric detection was carried out.

Partially purified bitter gourd ( Momordica charantia) peroxidase catalyzed decolorization of textile and other industrially important dyes

The aim of this study was to evaluate the enzymatic action of partially purified bitter gourd peroxidase for the degradation/decolorization of complex aromatic structures. Twenty-one dyes, with a wide spectrum of chemical groups, currently being used by the textile and other important industries have been selected for the study. Here, for the first time we have shown peroxidases from Momordica charantia (300 EU/gm of vegetable) to be highly effective in decolorizing industrially important dyes. Dye solutions, containing 50–200 mg dye/l, were used for the treatment with bitter gourd peroxidase (specific activity of 99.0 EU/mg protein). M. charantia peroxidases were able to decolorize most of the textile dyes by forming insoluble precipitate. When the textile dyes were treated with increasing concentration of enzyme, it was observed that greater fraction of the color was removed but four out of eight reactive dyes were recalcitrant to decolorization by bitter gourd peroxidase. Step-wise addition of enzyme to the decolorizing reaction mixture at the interval of 1 h further enhanced the dye decolorization. The rate of decolorization was enhanced when the dyes were incubated with fixed quantity of enzyme for increasing times. Decolorization of non-textile dyes resulted in the degradation and removal of dyes from the solution without any precipitate formation. Decolorization rate was drastically increased when the textile and other industrially important non-textile dyes were treated with bitter gourd peroxidase in presence of 1.0 mM 1-hydroxybenzotriazole. Complex mixtures of dyes were prepared by taking three to four reactive textile and non-textile dyes in equal proportions. Each mixture was decolorized by more than 80% when treated with the enzyme in presence of 1.0 mM 1-hydroxybenzotriazole. Our data suggest that the peroxidase/mediator system is an effective biocatalyst for the treatment of effluents containing recalcitrant dyes from textile, dye manufacturing, dyeing and printing industries.

Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH oxidase. Since it acts as a potent inhibitor in studies with neutrophils and macrophages, no inhibitory effect can often be found in non-phagocyte cells. In our experiments, apocynin even stimulated reactive oxygen species (ROS) production by vascular fibroblasts. Even when added to macrophages, apocynin initially caused an increase in ROS production. The inhibition of ROS formation followed, suggesting that in the presence of leukocyte myeloperoxidase and hydrogen peroxide, apocynin is converted to another compound. Apocynin pre-activated with H2O2 and horseradish peroxidase (HRP) inhibited ROS production immediately. In non-phagocytes, apocynin stimulated ROS production and no inhibition was observed even after 60 min. Apocynin treated with H2O2 and HRP, however, decreased ROS production in the same manner as in macrophages. The stimulatory effect on ROS production can be abolished by tiron and superoxide dismutase (SOD), suggesting that superoxide was the produced species. The effect of apocynin was inhibited by diphenylene iodinium (DPI), a non-scavenging NADPH oxidase inhibitor. It can be summarized that apocynin stimulates cell superoxide production. In the presence of peroxidase and hydrogen peroxide, however, it is converted into another compound that acts as an inhibitor of superoxide production. It strongly suggests that under conditions in vivo, apocynin can have opposite effects on phagocytes and non-phagocyte cells. It acts as an inhibitor of phagocyte NADPH oxidase but also as a ROS production stimulator in non-phagocyte cells.

A ready-to-use fluorimetric biosensor for superoxide radical using superoxide dismutase and peroxidase immobilized in sol–gel glasses

In this work, a highly sensitive fluorescent biosensor for quantitative superoxide radical detection, based on the coupled reaction superoxide dismutase–peroxidase enzymes and the use of the probe Amplex red, is described. Superoxide anion radical was produced via oxidation of xanthine by xanthine oxidase. Dismutation of superoxide was catalyzed by superoxide dismutase, generating hydrogen peroxide, which reacted stoichiometrically with the nonfluorescent Amplex red, in the presence of peroxidase, yielding the red-fluorescent oxidation product resorufin. The coupled superoxide dismutase–peroxidase system was immobilized in a single sol–gel matrix. The enzymatic activity of the encapsulated superoxide dismutase–peroxidase system was nearly identical to that of one of the soluble enzymes, indicating that sol–gel encapsulation preserved the hierarchy of the enzyme’s activity. Specificity and reusability of the encapsulated system for up to four cycles were also demonstrated. The fluorescent biosensor was able to detect concentrations of superoxide as low as 20 nM in phospholipid model membranes composed of saturated or unsaturated phospholipids. These facts make this biosensor a simple, reliable, and highly sensitive method with a potential use in biological systems, food, and drinks.

Kinetics and Thermodynamics of the Mediator Function of 9,10-Anthraquinone Derivatives in Electrochemical Reactions with Peroxidase

On the basis of the results of studying bioelectrocatalysis induced by electrochemical reductionof N-(9'10-anthraquinon-1-, -2-yl)oxamides under conditions close to physiological in the presence ofperoxidase, new criteria for estimation of the cholate-cholesterol coefficient of these compounds have beenproposed; these are the rate constants for homogeneous electron transfer from the reduced form of the quinone to the active center of peroxidase and thermodynamic parameters of the process.

Characterization of Superoxide-producing Sites in Isolated Brain Mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.

Peroxidase Substrate Nanosensors for MR Imaging

Magnetic nanoparticle conjugates that self-assemble in the presence of peroxidases were prepared as having large inducible magnetic resonance signals. When a serotonin−nanoparticle conjugate was incubated with myeloperoxidase, an enzyme implicated with inflammation and coronary diseases, there were significant changes in the T2 relaxation times of the solution, as measured by MRI. These nanoparticle conjugates can potentially serve as an in vivo diagnostic tool to identify peroxidase-induced diseases by MRI.

The characterization of peroxidases in mitochondria of maize roots

In the present study, we provide the evidence about the presence of peroxidases in mitochondria isolated from maize ( Zea mays L.) roots with characteristics of both, peroxidase and oxidase activities. Oxidase activity, mediating reduction of oxygen to superoxide anion (O 2 − ) and H 2O 2 with NAD(P)H as a substrate, in the presence of MnCl 2 and SHAM or p-coumaric acid, was demonstrated by using various known effectors and inhibitors of this reaction. Mitochondrial enzyme had some characteristics similar to MnCl 2/SHAM-stimulated NAD(P)H oxidase from cell wall and plasma membrane. Peroxidatic activity was detected as NAD(P)H oxidation in the presence of exogenously added H 2O 2 and various phenolic compounds. This peroxidase dependent NAD(P)H oxidation via phenolics had similar characteristics with guaiacol peroxidase from the same mitochondrial preparation. Different effects of Cu 2+, Fe 2+ and phenolic compounds on oxidase and peroxidase reactions suggest that these two activities are catalyzed by two separate, well-coordinated isoenzymes. Their role in generation and removal of reactive oxygen species (ROS) in plant cell was proposed.

Effect of cooking on oxalate content of pulses using an enzymatic procedure

An enzymatic method proposed for the determination of oxalate in urine is adapted for the estimation of soluble oxalate content in beans, chickpeas and lentils. Oxalates were extracted in water by refluxing for 2 h. The method is based on the oxidation of oxalate by the oxidase and the determination of the resulting hydrogen peroxide, which in presence of peroxidase, 3-methyl-2 benzotiazinolone and 3-dimethylamino benzoic, gives an indamine compound with an absorption maximum at 590 nm. The linearity (from 0.015 to 0.6 mM) of the method is adequate to the analysis of oxalate contents in pulses, and the inter-day precision of the method expressed as relative standard deviation was good (3.01%), with an accuracy of (98.7±2.5%) estimated by recovery assays. This method is applied to estimate the losses of soluble oxalate as a consequence of cooking. All the cooking procedures reduce the soluble oxalate content, but microwave and industrial procedures are more effective than traditional domestic cooking.

Idiosyncratic NSAID drug induced oxidative stress

Many idiosyncratic non-steroidal anti-inflammatory drugs (NSAIDs) cause GI, liver and bone marrow toxicity in some patients which results in GI bleeding/ulceration/fulminant hepatic failure/hepatitis or agranulocytosis/aplastic anemia. The toxic mechanisms proposed have been reviewed. Evidence is presented showing that idiosyncratic NSAID drugs form prooxidant radicals when metabolised by peroxidases known to be present in these tissues. Thus GSH, NADH and/or ascorbate were cooxidised by catalytic amounts of NSAIDs and hydrogen peroxide in the presence of peroxidase. During GSH and NADH cooxidation, oxygen uptake and activation occurred. Furthermore the formation of NSAID oxidation products was prevented during the cooxidation indicating that the cooxidation involved redox cycling of the first formed NSAID radical product. The order of prooxidant catalytic effectiveness of fenamate and arylacetic acid NSAIDs was mefenamic acid>tolfenamic acid>flufenamic acid, meclofenamic acid or diclofenac. Diphenylamine, a common moiety to all of these NSAIDs was a more active prooxidant for NADH and ascorbate cooxidation than these NSAIDs which suggests that oxidation of the NSAID diphenylamine moiety to a cation and/or nitroxide radical was responsible for the NSAID prooxidant activity. The order of catalytic effectiveness found for sulfonamide derivatives was sulfaphenazole>sulfisoxazole≫dapsone>sulfanilic acid>procainamide>sulfamethoxazole>sulfadiazine>sulfadimethoxine whereas sulfanilamide, sulfapyridine or nimesulide had no prooxidant activity. Although indomethacin had little prooxidant activity, its major in vivo metabolite, N-deschlorobenzoyl indomethacin had significant prooxidant activity. Aminoantipyrine the major in vivo metabolite of aminopyrine or dipyrone was also more prooxidant than the parent drugs. It is hypothesized that the NSAID radicals and/or the resulting oxidative stress initiates the cytotoxic processes leading to idiosyncratic toxicity.

An enzymatic assay for lysophosphatidylcholine concentration in human serum and plasma

Objectives: Several methods for measuring lysophosphatidylcholine (LPC) concentrations have been reported. However, these methods are not practical because they are either too complicated and/or too time-consuming for LPC determinations in human serum and plasma. Design and Methods: We have developed a new enzymatic LPC assay, which uses lysophospholipase, glycerophosphorylcholine phosphodiesterase and choline oxidase, and which determines the quantities of hydrogen peroxide generated in the presence of peroxidase using an oxidative chromogenic reagent and 4-aminoantipyrine. Results: Various samples were mixed with LPC assay reagents, and their changes in absorbance were measured. The present method produced a linear calibration line between LPC concentration and absorbance change. It also measured only LPC, and not other phospholipids such as phosphatidylcholine, sphingomyelin and lysophosphatidic acid. The within-run and between-run coefficients of variation were 0.3–0.7% and 0.7%, respectively. The recovery of exogenous LPC added to control serum was 99.5–102.1%. The correlation coefficient obtained in a comparison with a method for analyzing fatty acids was 0.9122. Conclusions: The present method is simple, specific for LPC, and can be applied with an automatic analyzer. It may also be useful for further studies of the biological functions of LPC as well as clinical applications in various disorders.

5-Fluoroindole-3-acetic acid: a prodrug activated by a peroxidase with potential for use in targeted cancer therapy

Indole-3-acetic acid and some derivatives are oxidized by horseradish peroxidase, forming a radical-cation that rapidly fragments (eliminating CO 2) to form cytotoxic products. No toxicity is seen when either indole-3-acetic acid or horseradish peroxidase is incubated alone at concentrations that together form potent cytotoxins. Unexpectedly, 5-fluoroindole-3-acetic acid, which is oxidized by horseradish peroxidase compound I 10-fold more slowly than indole-3-acetic acid, is much more cytotoxic towards V79 hamster fibroblasts in the presence of peroxidase than the unsubstituted indole. The fluorinated prodrug/peroxidase combination also shows potent cytotoxic activity in human and rodent tumor cell lines. Cytotoxicity is thought to arise in part from the formation of 3-methylene-2-oxindole (or analogues) that can conjugate with thiols and probably DNA or other biological nucleophiles. Levels of the fluorinated prodrug in the murine carcinoma NT after intraperitoneal administration of 50 mg/kg were about 200 μM. Although these were 4–5-fold lower than plasma levels (which reached 1 mM), the integrated area under the concentration/time curve in tumors over 2 hr was ∼20 mM min, almost double the exposure needed to achieve ∼90–99% cell kill in human MCF7 breast or HT29 colon tumor cell lines and CaNT murine cells in vitro, although the human bladder T24 carcinoma cell line was more resistant. The high cytotoxicity of 5-fluoroindole-3-acetic acid after oxidative activation suggests its further evaluation as a prodrug for targeted cancer therapy involving antibody-, polymer-, or gene-directed delivery of horseradish peroxidase or similar activating enzymes.

Microbial Transformations of p-Coumaric Acid by Bacillus megaterium and Curvularia lunata

p-Coumaric acid (1) is an abundant phenolic natural product that exhibits both chemoprotectant and antioxidant properties. Microbial transformation screening studies showed that 1 was converted to 4-vinylphenol (4), 4-hydroxybenzoic acid (2), caffeic acid (5), protocatechuic acid (6), and other unidentified metabolites over 144 h. 4-Vinylphenol (4) and its dimer, (2R,2S)-4-(2,3-dihydro-5-hyroxy-2-benzofuranyl) phenol (11), were produced by Bacillus megaterium, and 5-[(E)-2-carboxyethenyl]-2,3-dihydro-2-(4-hydroxyphenyl)-3-benzofurancarboxylic acid (15) and 4-hydroxybenzoic acid (2) were produced by Curvularia lunata. On the basis of deuterium-labeling experiments, B. megaterium catalyzes the nonoxidative enzymatic tautomerization of 1 to a vinylogous beta-keto acid intermediate that decarboxylates to 4. The presence of peroxidase and laccase activities in C. lunata extracts suggests that these enzymes may be involved in one-electron, p-coumaric acid dimerization in this organism.

Bilirubin Impairs Bactericidal Activity of Neutrophils through an Antioxidant Mechanism in Vitro

Background. Liver failure accompanied by hyperbilirubinemia after major hepatic resection is profoundly associated with septic complications. Although the immune dysfunction in cholestasis has been intensively investigated, the contribution of increased serum bilirubin to the impaired resistance to bacterial infection remains to be elucidated. Because bilirubin possesses an antioxidant activity, we hypothesized that bilirubin may scavenge reactive oxygen species (ROS) produced by neutrophils and consequently impair neutrophil bacterial killing. To address this, we evaluated the effects of bilirubin on the bactericidal activity of ROS or of neutrophils in vitro.Materials and methods. The antioxidant activity of bilirubin was determined using an ROS-sensitive fluorophore, dichlorofluorescin diacetate (DCFH-DA). Bilirubin concentration in the buffer solution was monitored spectorophotometrically after incubation with ROS. The effect of bilirubin on killing of Escherichia coli by ROS or by isolated human neutrophils was determined by counting the viable E. coli after incubation on nutrient agar.Results. The bilirubin concentration in the buffer solution was decreased by the addition of hydrogen peroxide, especially in the presence of peroxidase or ferrous iron. DCFH-DA oxidation by ROS or activated neutrophils was inhibited by bilirubin in a dose-dependent manner. The bactericidal activity of ROS or of isolated neutrophils was significantly attenuated by bilirubin.Conclusions. Bilirubin impairs bactericidal activity of neutrophils through scavenging ROS. Increased levels of serum bilirubin may well be responsible for the impaired bacterial clearance in patients with hyperbilirubinemia.

Rapid screening of H 2O 2 production by Mycoplasma mycoides and differentiation of European subsp. mycoides SC (small colony) isolates

Mycoplasma mycoides strains were screened for the ability to produce H 2O 2 from glucose and glycerol metabolism using rapid and simple colorimetric assays. In quantitative assays, H 2O 2 production by washed cell suspensions was detected by the oxidation of o-dianisidine in the presence of peroxidase. In qualitative assays, a 3,3′-diaminobenzidine-peroxidase reagent was applied to colonies on agar plates. Both methods enabled differentiation of European subsp. mycoides SC (small colony) isolates from other M. mycoides strains by their inability to produce H 2O 2 from glycerol metabolism. In addition, two strains of subsp. capri were identified which produced large amounts of H 2O 2 from glucose oxidation. In lysed cells of these strains, NADH oxidation gave approximately 1 mol H 2O 2 per mol NADH oxidised whereas in 36 subsp. mycoides and 10 other subsp. capri strains, the quantity produced was 0.01–0.20 mol H 2O 2 per mol NADH oxidised.