Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes

The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD = 5.0 pmol/assay and LLQ = 30 pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09 nmol hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31 nmol of hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.     

A model of peroxidase activity with inhibition by hydrogen peroxide

The rate of color formation in an activity assay consisting of phenol and hydrogen peroxide as substrates and 4-aminoantipyrine as chromogen is significantly influenced by hydrogen peroxide concentration due to its inhibitory effect on catalytic activity. A steady-state kinetic model describing the dependence of peroxidase activity on hydrogen peroxide concentration is presented. The model was tested for its application to soybean peroxidase (SBP) and horseradish peroxidase (HRP) reactions based on experimental data which were measured using simple spectrophotometric techniques. The model successfully describes the dependence of enzyme activity for SBP and HRP over a wide range of hydrogen peroxide concentrations. Model parameters may be used to compare the rate of substrate utilization for different peroxidases as well as their susceptibility to compound III formation. The model indicates that SBP tends to form more compound III and is catalytically slower than HRP during the oxidation of phenol.     

A rapid assay for peroxidase activity

1. Peroxidase has been assayed by a chronometric method involving the coupled reaction of ascorbic acid with the product of the enzymic action on benzidine. 2. Measurements of the activities of horseradish and tea peroxidase by this and two other methods, involving respectively pyrogallol and o-dianisidine, are compared. 3. It is claimed that the chronometric method is relatively simple, rapid and accurate. 4. The method can be used in the presence of polyphenol oxidases.     

Organospecific responses of lupin seedlings to lead Localization of hydrogen peroxide and peroxidase activity

In an earlier work using tissue printing method, we found that the PR-10 stress protein was observed in leaf petiole of lupin seedling where lead was not detected (Przymusiński et al. 2001). These results suggested the presence of substance(s) mediating a signal transduction from directly affected cells to distant organs. As the hydrogen peroxide was found to be involved in signal transduction pathway, in the present paper, we analysed the level of H₂O₂ in the organ of lupin seedlings exposed to Pb²⁺ with spectrophotometric method and tissue printing technique. It was unequivocally demonstrated that the level of H₂O₂ and the activity of peroxidase increased in every tested organ of lead-treated lupin seedling. Both the level of H₂O₂ and the activity of POX were correlated with amount of Pb²⁺ ions in the cells (Przymusiński et al. 2001) and decreased in tissues more and more distant from the site of metal application. On the other hand, there was no correlation between the histological localization of H₂O₂ and peroxidase. Our results seem to confirm the hypothesis that H₂O₂ may act as a signalling substance involved in the induction of PR protein synthesis. It was indicated that there is high degree of correlation between the localization of H₂O₂ and the histological localization of PR-10 proteins (Przymusiński et al. 2001) in every tested organ of lupin seedling. The presented hypothesis is also supported by the fact that H₂O₂ and PR-10 proteins are detected in organs and tissues where Pb²⁺ was not found at all.     

Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol-aniline coupling hydrogen donor

The hydrogen donor couples pyrocatechol-aniline and phenol-aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV-Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol-aniline was converted to a pink-colored reagent with a lambda(max) of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol-aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol-aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of K(m) (12.5mM) and V(max) (12.2 mM min(-1)mg(-1)) were calculated for pyrocatechol-aniline. Regarding the superiority of pyrocatechol-aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay.     

A new assay to measure RNA N-glycosidase activity

We have developed a convenient procedure to measure the activity of Ricin A-chain and other enzymes with RNA N-glycosidase activity. The method is based on the use of a tritiated oligoribonucleotide as a substrate. The enzymatic activity is directly determined by measuring the release of adenine from the substrate. This method should prove useful in the study of the molecular mechanism of action of Ricin A and other RNA N-glycosidases.     

Ethylene-promoted ascorbate peroxidase activity protects plants against hydrogen peroxide, ozone and paraquat

Abstract. In experiments where mung beans ( Vigna radiata L.) and peas ( Pisum sativum L.) have been pre-exposed to ethylene and afterwards treated with ozone, it has been shown that such ethylenepretreated plants may become more resistant to ozone. Further experiments with hydrogen peroxide (H₂O₂) and the herbicide paraquat suggest that this increased resistance against ozone depends on the stimulation of ascorbate peroxidase activity which provides cells with increased resistance against the formation of H₂O₂ which is also formed when plants are fumigated with ozone. These results explain why increased production of ethylene can be observed in plants exposed with ozone or other oxidative stress and clearly demonstrate that in plants, as well as animals, peroxidases protect cells against harmful concentrations of hydroperoxides.     

Horseradish peroxidase inhibition and antioxidant activity of ebselen and related organoselenium compounds

Horseradish peroxidase (HRP) inhibition and glutathione peroxidase (GPx) activities of ebselen and some related derivatives are described. These studies show that ebselen and ebselen ditelluride (EbTe(2)) with significant antioxidant activity, inhibit the HRP-catalyzed oxidation reactions. In addition, inhibition of lipid peroxidation and singlet oxygen quenching studies were carried out. Although the inhibition of HRP by ebselen is comparable with that of EbTe(2), the inhibitory effect on gamma-radiation induced lipid peroxidation and the GPx activity of ebselen is found to be much higher than that of EbTe(2).     

A simple cell based assay to measure Parkin activity

Parkin is an ubiquitin-protein ligase mutated in Autosomal Recessive - Juvenile Parkinsonism. Here, we describe a cell-based assay to measure Parkin's ubiquitin-protein ligase activity. It relies on the ability of Parkin to recognise depolarised mitochondria and exploits a cell line where Parkin expression is inducible. In these cells, Parkin expression promotes mitophagy and accelerates cell death in response to mitochondrial depolarisers. Time-lapse imaging confirmed cell death and revealed increased perinuclear mitochondrial clustering following induction of Parkin expression in cells exposed to carbonyl cyanide m-chlorophenylhydrazone. Similar effects were not observed with α-synuclein or DJ-1, other proteins associated with the development of Parkinson's disease, confirming the specificity of the assay. We have used this assay to demonstrate that ligase-defective Parkin mutants are inactive, and cellular proteasomal activity (using the proteasomal inhibitors MG132, clasto-lactacystin β-lactone and epoxomicin) is essential for the Parkin mediated effect. As the assay is suitable for high-throughput screening, it has the potential to identify novel proteostasis compounds that stimulate the activity of Parkin mutants for therapeutic purposes, to identify modulators of kinase activities that impact on Parkin function, and to act as a functional read-out in reverse genetics screens aimed at identifying modifiers of Parkin function during mitophagy.     

A sensitive radiometric assay to measure D-xylulose kinase activity

A radiometric test system for D-xylulose kinase (XK) was developed for the measurement of enzyme activity in crude cell extracts and to minimize the volume of reaction mixtures besides increasing the sensitivity. [U-14C]xylulose 5-phosphate was produced from commercially available [U-14C]xylose in a coupled assay system containing D-xylose isomerase, which yields [U-14C]xylulose, the substrate of ATP-dependent D-xylulose kinase. Separation of products and substrates was achieved by thin layer chromatography, identification of radioactive spots by radioscanning followed by quantitative scintillation counting. The protocol was validated through determination of kinetic constants of a purified His-tagged enzyme from Escherichia coli and comparison with the spectrophotometric method. The radiometric assay was applied to determine xylulose kinase activity in crude cell extracts from a variety of eukaryotic and prokaryotic organisms.     

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.     

The scopoletin assay for hydrogen peroxide. A review and a better method

Scopoletin, 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, a naturally occurring component in cotton leaf and citrus peel is a fluorescent substrate for peroxidase which has been used by many investigators for the determination of hydrogen peroxide concentration. The technical details of these investigations are application-specific and rather critical, making it difficult to apply the scopoletin assay to alternative systems without extensive modification. Although such factors as interfering substances and optimum conditions have been discussed in many publications, these discussions tend to be application-specific. The present paper attempts to provide a technical review of scopoletin applications, add a few new experimental observations, and discuss general parameters which must be carefully controlled for reliable results.     

The effect of chitooligosaccharides on hydrogen peroxide production and anionic peroxidase activity in wheat coleoptiles

We studied the effects of chitooligosaccharides (ChOS) with a mol wt of 5 kD, the degree of acetylation of 65%, and the concentrations from 0.01 to 100 mg/l on the content of hydrogen peroxide in incubation medium and the activity of anionic peroxidase (pI 3.5) in the segments of wheat (Triticum aestivum) coleoptiles. H₂O₂ production and peroxidase activity were found to be dependent on the ChOS concentration. After 3 h of incubation, the highest H₂O₂ level in medium was observed at 0.01 mg/l ChOS, whereas after 6h, at 1 mg/l. After 3 h of incubation, ChOS suppressed peroxidase activity. After 6 h of incubation, high ChOS concentrations enhanced peroxidase activity. IAA favored H₂O₂ accumulation in medium and suppressed anionic peroxidase. The involvement of ChOS in the control of the level of reactive oxygen species and anionic peroxidase activity in plant cells is suggested.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 238–242.Original Russian Text Copyright © 2005 by Yusupova, Akhmetova, Khairullin, Maksimov.This revised version was published online in April 2005 with a corrected cover date.     

Cell wall peroxidase activity,hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings

The changes in cell-wall peroxidase (POD) activity and H₂O₂ level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively reduced root growth and increased ionically bound cell-wall POD activity. NaCl had no effect on covalently bound cell-wall POD activities. The reduction of root growth by NaCl is closely correlated with the increase in H₂O₂ level. Exogenous H₂O₂ was found to inhibit root growth of rice seedlings. Since ammonium and proline accumulation are associated with root growth inhibition caused by NaCl, we determined the effects of NH₄Cl or proline on root growth, cell-wall POD activity and H₂O₂level in roots. External application of NH₄Cl or proline markedly inhibited root growth, increased cell-wall POD activity and increased H₂O₂ level in roots of rice seedlings in the absence of NaCl. An increase in cell-wall POD activity and H₂O₂ level preceded inhibition of root growth caused by NaCl, NH₄Cl or proline. NaCl or proline treatment also increased NADH-POD and diamine oxidase (DAO) activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the H₂O₂ generation in the cell wall of NaCl- or proline-treated roots. NH₄Cl treatment increased NADH-POD activity but had no effect on DAO activity, suggesting that NADH-POD but not DAO is responsible for H₂O₂ generation in cell wall of NH₄Cl-treated roots.     

A novel and simple route to prepare a Pt nanoparticle-loaded carbon nanofiber electrode for hydrogen peroxide sensing

A facile wet-chemical method was developed to prepare a novel Pt nanoparticle-loaded carbon nanofiber (Pt/CNF) electrode. Without using any stabilizer or pretreatment procedure, large amounts of Pt nanoparticles could be well deposited on the surface of the electrospun CNF electrode at room temperature, as revealed by scanning electron microscopy (SEM). The effect of the precursor concentration on the formation of Pt catalysts was investigated to optimize the performance of the proposed hybrid electrode. When applied to the electrochemical detection of hydrogen peroxide (H?O?), the Pt/CNF electrode exhibited low overpotential, fast response and high sensitivity. A low detection limit of 0.6 μM with wide linear range of 1-800 μM (R=0.9991) was achieved at the Pt/CNF electrode, which was superior to that obtained with other H?O? electrochemical sensors reported previously. In addition, the Pt/CNF electrode showed good selectivity for H?O? detection in the presence of ascorbic acid (AA), acetaminophenol (AP) and uric acid (UA) under physiological pH condition. The attractive analytical performances and facile preparation method made this novel hybrid electrode promising for the development of effective H?O? sensors.     

Reaction of hydrogen peroxide and peroxidase activity in carboxymethylated cytochrome c: spectroscopic and kinetic studies

The peroxidase activity of carboxymethylated cytochrome c (Cmcytc) has been investigated by spectroscopic and kinetic techniques to examine the effect of carboxymethylation on the peroxidase activity of native cytochrome c (cytc). The optical spectrum suggests that the reaction of Cmcytc with H(2)O(2) proceeds through only one intermediate, compound I. The apparent rate constant (k(app)) for the reaction was found to be 17, 72 and 210 M(-1) s(-1) at pH 7.0, 5.0 and 3.5 respectively. These values are about 60 times larger than those reported for native cytc (0.236 M(-1) s(-1) at pH 7.0), and about five orders of magnitude lower than those for classical peroxidases. Cmcytc was found to catalyse oxidation of organic and inorganic substrates. The second order rate constant for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) by Cmcytc (205 [H(2)O(2)] s(-1)) is found to be larger than the corresponding value for native cytc (50 [H(2)O(2)] s(-1)) at pH 6.0. The carboxymethylation of cytc ruptures the Fe-S (Met 80) bond and increases the rate of its reaction with H(2)O(2), and its catalytic activity. The specific activity of Cmcytc was measured spectrophotometrically by the reported method using ABTS as substrate, and was found to be 288, 473 and 872 microM min(-1) mg(-1) at pH 7.0, 5.0 and 3.5 respectively. Resonance Raman studies indicated the presence of a bis-histidine coordinated form of Cmcytc at neutral pH, and the existence of a population distribution of different ligation states such as bis-histidine (HH), histidine-water (HW) and five coordinate (5C) forms at lower pH. The relative population of different species in Cmcytc was found to be HH (approximately 100%, approximately 50%, approximately 44%), HW (approximately 0%, approximately 44%, 41%) and 5C (approximately 0%, approximately 6%, 15%) at pH 7.0, 4.7 and 3.1 respectively. We have attempted to correlate the pH dependence of the reaction of Cmcytc with hydrogen peroxide and its peroxidase activity with the haem stereochemical structures observed for Cmcytc. Steady-state and time-resolved tryptophan fluorescence studies on Cmcytc were done to probe the conformational changes around the haem pocket of Cmcytc.     

A kinetic study on the suicide inactivation of peroxidase by hydrogen peroxide

In the absence of reductant substrates, and with excess H2O2, peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) shows the kinetic behaviour of a suicide inactivation, H2O2 being the suicide substrate. From the complex (compound I-H2O2), a competition is established between two catalytic pathways (the catalase pathway and the compound III-forming pathway), and the suicide inactivation pathway (formation of inactive enzyme). A kinetic analysis of this system allows us to obtain a value for the inactivation constant, ki = (3.92 +/- 0.06) x 10(-3) x s-1. Two partition ratios (r), defined as the number of turnovers given by one mol of enzyme before its inactivation, can be calculated: (a) one for the catalase pathway, rc = 449 +/- 47; (b) the other for the compound III-forming pathway, rCoIII = 2.00 +/- 0.07. Thus, the catalase activity of the enzyme and, also, the protective role of compound III against an H2O2-dependent peroxidase inactivation are both shown to be important.