The mechanism of indoleacetic acid oxidase reaction catalyzed by turnip peroxidase.
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Brassica napus↗
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Results for “OXIDASE AND PEROXIDASE”
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A comparison between the NADPH oxidase activity of human polymorphonuclear leukocytes and the oxidase activity of several purified peroxidases.
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Ascorbic Acid↗
Hepatic microsomal N-hydroxylation of adenine to 6-N-hydroxylaminopurine.
The enzymatic N-hydroxylation of the purine base adenine to the genotoxic and mutagenic compound 6-N-hydroxylaminopurine is reported for the first time. Adenine was N-oxygenated in vitro by aerobic incubations with 3-methylcholanthrene or isosafrole induced microsomal fractions of rat liver homogenates and NADPH. The formation of 6-N-hydroxylaminopurine in the incubation mixtures under widely differing conditions was assayed using newly-developed, high-performance liquid- and thin-layer chromatographic methods. Optimal reaction conditions and kinetic parameters were determined. Neither superoxide anion nor hydrogen peroxide was directly involved in the N-hydroxylation reaction. Oxidases like xanthine oxidase and peroxidase (in the presence of hydrogen peroxide) did not catalyse this N-hydroxylation. The involvement of cytochrome P-450 isoenzymes in this reaction is supported by the observation that the N-hydroxylation is only observed after pretreatment of the rats with 3-methylcholanthrene or isosafrole. Other inducers (phenobarbital, ethanol, 5-pregnen-3 beta ol-20-one-16 alpha-carbonitrile) were without effect. This is the first example of the microsomal transformation of an endogenous substance to a toxic derivative by usually foreign substances (xenobiotics) metabolizing cytochrome P-450 isoenzymes. The significance for the in vivo situation is discussed on the basis of the data obtained in this study.
Adenine↗
Hydroxylation; the activation of oxygen by peroxidase.
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Coloring Agents↗
Molecular interactions in the phenomenology of the onset of mental illness.
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Acetylcholine↗
Mechanism(s) for the metabolism of mitoxantrone: electron spin resonance and electrochemical studies.
Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (-0.79 V) when compared to diaziquone (-0.36 V) and Adriamycin (-0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.
Biotransformation↗
A new colorimetric method for the determination of plasma lecithin-cholesterol acyltransferase activity.
A new method has been developed to improve the linearity of the plasma lecithin-cholesterol acyltransferase reaction by adding synthetic dipalmitoyl lecithin sol to the incubation medium and subsequently measuring the change in free cholesterol content after incubation by a combined enzymatic method using cholesterol oxidase and peroxidase. The method was found to be simpler than other techniques such as gas-liquid chromatography or isotope methods and therefore may be suitable for the routine assays of plasma lecithin-cholesterol acyltransferase activities in clinical laboratories.
Acyltransferases↗
Determination of free and esterified cholesterol by a kinetic method. II. Evaluation of the enzymic method which uses 2,2-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS) as chromogen.
A simple kinetic method for the determination of free and esterified serum cholesterol based on the oxidation of 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS) by use of choilesterol esterase, cholesterol oxidase and peroxidase has already been reported. Here the method is statistically examined. The method is very sensitive and precise (C.V. below 5%). The standard curve is linear up to 25.9 mmol/l. Comparison with results by Abell's method gave a linear regression of Yx = 0.2025 + 1.0043X with a correlation coefficient (r) of 0.983. Comparison with the enzymic methods of Roeschlau et al., Trinder, and Allain et al. gave Yx = 0.2037 + 0.9549X (r = 0.975), Yx = 0.4777 +0.8857X (r = 0.958) and Yx = 0.244 + 0.932X (r = 0.970), respectively. The effects of haemoglobin and bilirubin were studied and normal ranges for the method were determined on 150 healthy mature subjects of both sexes between 20 and 45 years of age. They are 3.375 to 6.948 mmol/l for total cholesterol, and 0.7196 to 2.089 mmol/l for free cholesterol.
Adult↗
A rapid method for measurement of ethylene glycol.
It has been reported that ethylene glycol produces a positive interference in the triglyceride assay on the DuPont aca discrete analyzer. The sensitivity of this method for ethylene glycol was exploited to develop a rapid and convenient method for detecting and quantitating ethylene glycol in serum by the use of a "triglyceride gap." This method is based on the difference between two different enzymatic triglyceride measurements; the DuPont aca triglyceride measurement, which uses lipase and glycerol dehydrogenase, and a Boehringer Mannheim method, which uses lipase, glycerol kinase, glycerolphosphate oxidase, and peroxidase. Serum pools were spiked with increasing concentrations of ethylene glycol to construct a standard curve (linear to 25 mmol/L). Patients specimens and control sera were analyzed using a one point calibration. Within-run and between-run coefficients of variation (CV) of 1.5, 2.8, 5.8, and 3.2%; 3.4%; and 12.6% were obtained at ethylene glycol concentrations of 20.13, 8.37, and 2.18 mmol/L, respectively. The sensitivity of this method is 1.0 mmol/L. Methanol, ethanol, n-propanol, isopropanol, and acetone at 100 mmol/L; 20 mmol/L 1,3-propanediol; and 10 mmol/L glycolic acid, oxalic acid, glyoxylic acid, and lactic acid did not interfere in the quantitation of ethylene glycol. High levels (10 mmol/L) of beta-hydroxybutyrate and glycerol showed a slight positive interference on the quantitation of ethylene glycol, whereas 10 mmol/L glycoaldehyde caused a substantial overestimation of serum ethylene glycol. The presence of propylene glycol, at levels ranging from 5 to 50 mmol/L, resulted in an underestimation of serum ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)
Artifacts↗
Caracterisations immunochimiques de deux auxine oxydases extraites de tumeurs vegetables.
Two auxin oxidases with distinct antigenic specificities have been identified in extracts of plant tumor induced in Datura stramonium by Agrobacterium tumefaciens. Auxin oxidase and peroxidase activities are demonstrated for both enzymes. The adaptation of an immunochemical method allows the quantitative evaluation of both enzymes in different plant tissue extracts. The amount of these enzymes which is low in the organ of the healthy plant increases during tumorisation, healing and in vitro culture of healthy tissues.
Journal Article↗
Quantitation of proteins by dot-immunobinding assay. A comparison of visualization methods using eukaryotic initiation factor 2 and a monospecific antibody.
Various visualization methods were compared for quantitation of proteins by the dot-immunobinding assay. Comparisons were carried out using a multi-subunit protein, eukaryotic initiation factor 2, and monospecific antibodies directed against two of the factor's subunits. The protein was spotted onto nitrocellulose and the membranes were incubated with primary antibody. The antigen-antibody complex was visualized by one of six methods using either alkaline phosphatase-, horseradish peroxidase-, or glucose oxidase-conjugated IgG, or colloidal gold-labelled IgG, colloidal gold-labelled IgG with silver enhancement, or 125I-labelled protein A. The amount of secondary antibody bound was quantitated by densitometric scanning of the nitrocellulose membrane after staining or autoradiography. The sensitivity of each of the methods was similar; each of the visualization methods could detect less than 1 ng of protein by the dot-immunobinding assay. Curves of protein concentration vs. densitometric absorbance were found to fit a parabolic relationship (r2 = 0.99) over a wide range of concentrations.
Alkaline Phosphatase↗
A continuous spectrophotometric method for monitoring phospholipase D-catalyzed reactions of physiological substrates.
A continuous spectrophotometric method for monitoring phospholipase D-catalyzed hydrolysis of long acyl chain phosphatidylcholines has been formulated at pH 8.0 in a mixed detergent system using the coupling enzymes choline oxidase and peroxidase. Standard curves for phosphatidylcholine determination in both end-point and rate modes are presented and applied to the estimation of that phospholipid in a solubilized human erythrocyte membrane sample. In rate mode the method is suitable for kinetic study of phospholipase D with physiological substrates in micellar form.
Catalysis↗
Sensitive method for HPLC determination of acetylcholine, choline and their analogues using fluorometric detection.
A method is described for reversed-phase HPLC separation of acetylcholine and choline and of their homologues in tissue extracts or perfusion fluids, combined with postcolumn enzymatic derivatization and fluorometric quantification. The separation occurs on a polymeric resin derivatized with hydrophobic moiety and the mobile phase consists of Na2HPO4, 3-(p-hydroxyphenyl)propionic acid and sodium dodecylsulphate; postcolumn enzyme reactor contains immobilized acetylcholinesterase, choline oxidase, and peroxidase. The limits of detection are 1 pmol choline and 3 pmol acetylcholine per sample. The method is free of interferences encountered with electrochemical detection and well suited for non-attended automatic operation.
Acetylcholine↗
Glucose potentiometric electrodes based on mediatorless bioelectrocatalysis. A new approach.
A potentiometric method has been developed for analysis of glucose on the basis of glucose oxidase and peroxidase co-immobilized on the surface of an electrode made of composite carbonic material. Catalytic oxidation of glucose results in the formation of hydrogen peroxide. Mediatorless peroxidase catalysis of electroreduction of hydrogen peroxide leads to a shift in the electrode potential. The rate of the increase of the electrode potential is proportional to glucose concentration and calibration curve is linear at 0.025-2.0 mM. Electrodes permit at least 100 measurements without any loss in the activity. Electrodes remain stable for 90 days when stored at 277 K.
Biosensing Techniques↗
Potentiometric biosensors for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis.
A potentiometric method for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis has been developed. The method includes coimmobilization of three enzymes, butyrylcholinesterase, choline oxidase and peroxidase, on composite carbon electrodes. Catalytic hydrolysis of butyrylcholine and subsequent catalytic oxidation of choline result in the formation of hydrogen peroxide leads to a shift in the electrode potential. The detection limit for trichlorfon analysis is 2 x 10(-13) M. Electrodes remain stable for at least 4 weeks when stored at 277 K.
Biosensing Techniques↗
Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4.
A fundamental aspect of cytochrome P450 function is the role of the strictly conserved axial cysteine ligand, replacement of which by histidine has invariably resulted in mammalian and bacterial preparations devoid of heme. Isolation of the His-436 variant of NH2-truncated P450 2B4 partly as the holoenzyme was achieved in the present study by mutagenesis of the I-helix Ala-298 residue to Glu and subsequent conversion of the axial Cys-436 to His. The expressed A298E/C436H double mutant, cloned with a hexahistidine tag, had a molecular mass equivalent to that of the primary structure of His-tagged truncated 2B4 and the sum of the two mutated residues, and contained a heme group which, when released on HPLC, showed a retention time and spectrum identical to those of iron protoporphyrin IX. The absolute spectra of A298E/C436H indicate a change in heme coordination structure from low- to high-spin, and, as expected for a His-ligated hemeprotein, the Soret maximum of the ferrous CO complex is at 422 nm. The double mutant has no oxygenase activity with representative substrates known to undergo transformation by the oxene [(FeO)3+] or peroxo activated oxygen species, but catalyzes significant H2O2 formation that is NADPH- and time-dependent, and directly proportional to the concentration of A298E/C436H in the presence of saturating reductase. Moreover, the catalytic efficiency of A298E/C436H in the H2O2-supported peroxidation of pyrogallol is more than two orders of magnitude greater than that of wild-type 2B4 or the A298E variant. The results unambiguously demonstrate that the proximal thiolate ligand is essential for substrate oxygenation by P450.
Amino Acid Substitution↗
Simultaneous detection of L-glutamate and nitric oxide from adherently growing cells at known distance using disk shaped dual electrodes.
An ex vivo system for simultaneous detection of nitric oxide (NO) and L-glutamate using integrated dual 250 microm platinum disk electrodes modified individually with suitable sensing chemistries has been developed. One of the sensors was coated with an electrocatalytic layer of Ni tetrasulfonate phthalocyanine tetrasodium salt (Ni-TSPc) covered by second layer of Nafion, which stabilises on the one hand the primary oxidation product NO(+) and prevents interferences from negatively charged compounds such as NO(2)(-). For glutamate determination, the second electrode was modified with a crosslinked redox hydrogel consisting of Os complex modified poly(vinylimidazol), glutamate oxidase and peroxidase. A manual x-y-z micromanipulator on top of an inverted optical microscope was used to position the dual electrode sensor at a defined distance of 5 microm from a cell population under visual control. C6 glioma cells were stimulated simultaneously with bradykinin or VEGF to release NO while KCl was used to invoke glutamate release. For evaluation of the glutamate sensors, in some experiments HN10 cells were used. To investigate the sensitivity and reliability of the system, several drugs were applied to the cells, e.g. Ca(2+)-channel inhibitors for testing Ca(2+)-dependence of the release of NO and glutamate, rotenone for inducing oxidative stress and glutamate antagonists for analysing glutamate release. With these drugs the NO and glutamate release was modulated in a similar way then expected from previously described systems or even in-vivo measurements. We therefore conclude that our system is suitable to analyse stress-induced mechanisms in cell lines.
Animals↗
Enzymatic microreactors in chemical analysis and kinetic studies.
The fields of application of microreactors are becoming wider every year. A considerable number of papers have been published recently reporting successful application of enzymatic microreactors in chemistry and biochemistry. Most are devices with enzymes immobilized on beads or walls of microfluidic channels, whilst some use dissolved enzymes to run a reaction in the microfluidic system. Apart from model systems, mostly with glucose oxidase, horseradish peroxidase and alkaline phosphatase, the principal fields of application of microreactors are tryptic digestion of proteins and polymerase chain reaction in automated analyses of proteomic and genetic material, respectively. Enzymatic microreactors also facilitate characterization of enzyme activity as a function of substrate concentration, and enable fast screening of new biocatalysts and their substrates. They may constitute key parts of lab-on-a-chip and muTAS, assisting the analysis of biomolecules. This review provides systematic coverage of examples of reports on enzymatic microreactors published recently, as well as relevant older papers.
Bioreactors↗