Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.11.1.7 (peroxidase)
65,474 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A chemiluminescent flow-sensing device for the determination of phospholipase D (PLD) activity and/or choline (Ch) in biological samples using choline oxidase (ChO) and horseradish peroxidase (HRP) immobilized on Eupergit C (polymer beads of methacrylamide, N-methylene-bis-methacrylamide, and allyl-glycidyl-ether) was developed. The best results were obtained with immobilized ChO and HRP at a polymer beads wet weight ratio of 16:1. The optimized parameters of the developed sensing device were 56 microM luminol in working solution; sample volume, 60 microliters; flow rate, 0.3 ml/min; and sample throughput, 15/h. The detection limit (3 SD) using a luminescent enhancer was 1.2 microM for Ch, corresponding to 0.167 mIU of PLD activity per milliliter. Without enhancer the values were 3.0 microM and 0.417 mIU, respectively. The Ch recovery varied between 80.4 and 109%. The biological samples quenched the luminescent light to different extents, and this matrix effect was readily overcome by measuring the luminescent signal of added Ch standard. The flow biosensor was used for the determination of PLD in samples of different origin, including rape seeds during maturation.
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PMID:A chemiluminescent flow sensing device for determination of choline and phospholipase D activity in biological samples. 905 96

A chemiluminescent system has been developed for ultrasensitive, quantitative analysis as well as visualization of the spatial distribution of biomolecules such as antigens, enzymes, antibodies, DNA probes in tissue, or cells. The system consists of a low-light imaging Vidicon videocamera connected to an optical microscope, able to measure light at the single photon level and perform 3D image analysis of the subcellular distribution of the analyte. The concentration and the spatial distribution of enzymes, or enzyme-labeled biospecific reagents can be determined using appropriate chemiluminescent substrates. Analytes are also determined with coupled enzymatic reactions terminating in light emission. Oxirane acrylic beads (250-micron-diameter macroporous particles) with immobilized horseradish peroxidase have been used as a model system to optimize the experimental conditions in terms of signal intensity and spatial resolution as a function of different chemiluminescent substrates such as luminol/enhancer/H2O2 and acridancarboxylate ester/H2O2. Localization of oxirane beads immobilized acetylcholinesterase has been also used to optimize a system in which the detection and localization of the primary enzyme involves two secondary enzymes in solution, choline oxidase and horseradish peroxidase, leading to a final light emission. Immunoenzymatic reactions for the detection of viral antigens and in situ hybridization assays for the detection of viral DNAs (cytomegalovirus, herpes simplex virus) have been performed in cells using peroxidase-labeled antibodies or cDNA probes and the analytical performance of different chemiluminescent substrates for the enzyme has been evaluated. The results obtained showed the possibility to sharply image the bioprobes in single cells and peroxidase is a suitable label when luminol/H2O2 system is used in conjunction with enhancer as in the ECL and SuperSignal Ultra reagents; other substrates such as Lumigen PS-3, despite adequate detectability, showed problems of localization of the signal as a result of the relatively long half-life of the excited emitting species and its diffusion in the chemiluminescent cocktail. The system has proven to be highly sensitive, able to perform quantitative analysis, and relatively simple.
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PMID:Chemiluminescent imaging of enzyme-labeled probes using an optical microscope-videocamera luminograph. 951 72

An on-line acetylcholine (ACh) sensor was developed in order to determine extracellular ACh concentrations without interference from choline (Ch). The sensor is composed of a small-volume enzymatic prereactor (22-microL inner volume) in which choline oxidase and catalase are immobilized in series. Carbon electrodes were modified with an acetylcholine esterase (AChE), choline oxidase (ChOx), and osmium poly(vinylpyridine)-based redox polymer containing horseradish peroxidase (Os-gel-HRP). The sensor sensitivity was 43.7 nA/microM (+/- 0.15, n = 3) for ACh under optimized conditions. Almost no response was seen when 100 microM Ch was continuously injected. The detection limit for ACh with the sensor was comparable to that obtained using liquid chromatography with electrochemical detection combined with an enzymatic reactor. The electrical stimulation of cultured rat hippocampal tissue resulted in an extracellular ACh increase of 20 nM (+/- 11 nM, n = 3). This increase was observed continuously with our online sensor combined with a microcapillary sampling probe located very close to the tissue. The continuous measurement of ACh and Ch using a split disk carbon film dual electrode in which one electrode surface was modified with ChOx/Os-gel-HRP and the other with AChE-ChOx/Os-gel-HRP bilayer film was also demonstrated to improve the response time by eliminating the prereactor.
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PMID:On-line electrochemical sensor for selective continuous measurement of acetylcholine in cultured brain tissue. 953 3

Disposable screen-printed, film carbon electrodes (PFCE) were modified with cast-coated Osmium-polyvinylpyrridine-wired horse radish peroxidase gel polymer (Os-gel-HRP) to enable the detection of the reduction at 0 mV of hydrogen peroxide (H2O2) derived from a post-column immobilized enzyme reactor (IMER) containing acetylcholinesterase and choline oxidase. In another series of experiments PFCE were initially modified with cast-coated Os-gel-HRP and then treated with glucose oxidase in bovine serum albumin (BSA) and cross-linked with glutaraldehyde to form a bi-layer glucose-Os-gel-HRP PFCE. This bi-layer glucose-Os-gel-HRP PFCE generated a reduction current at 0 mV to H2O2 derived from the reaction of glucose oxidase and glucose in solution. These enzyme-modified PFCE were housed in a radial flow cell and coupled with cation-exchange liquid chromatographic methods to temporally separate substrates in solution for the determination of acetylcholine (ACh) and choline (Ch) in the first experimental series, or glucose in the second experimental series. These two disposable enzyme-modified PFCE exhibited linear current vs. substrate relations, were durable, being usable for approximately 40 determinations, and were sufficiently sensitive to be employed in biological sampling. Both assays utilized the same HPLC equipment. The limit of detection for ACh was 16 fmol/10 microl and that for glucose was 12 micromol/7.5 microl. ACh and Ch were measured from a microdialysate from the frontal cortex of a rat. Glucose in human urine was determined using the bi-layer glucose oxidase-Os-gel-HRP PFCE.
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PMID:Disposable, enzymatically modified printed film carbon electrodes for use in the high-performance liquid chromatographic-electrochemical detection of glucose or hydrogen peroxide from immobilized enzyme reactors. 961 27

Potentiometric choline electrodes were developed on the basis of the mediator-free bioelectrocatalysis. The electrodes made of a composite carbon-polymer material contain choline oxidase and peroxidase coimmobilized on the surface of the electrode. The rate of the potential increase was shown to be proportional to the choline concentration within a broad range of variation. Coupling of choline-sensitive electrodes with butyrylcholinesterase makes possible both the direct detection of butyrylcholine and analysis of butyrylcholinesterase inhibitors.
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PMID:[Potentiometric electrodes for determining choline, butyrylcholine and cholinesterase inhibitors]. 964 13

A collaborative study was conducted on a coupled enzymatic-spectrophotometric method for the determination of choline in infant formula and milk powders. Twenty-nine laboratories participated in the analysis of 8 blind duplicates over the range of 45-175 micrograms/100 g sample. After the combined acid hydrolysis-phospholipase release of choline, incubation with choline oxidase in the presence of peroxidase and phenol generates a quinoneimine chromophore with 4-aminoantipyrine. Following raw data screening, overall mean RSDR was estimated at 5.14% (range, 4.26-6.34%) with a HORRAT value of 0.91 (range, 0.76-1.00) and an overall mean RSDr:RSDR value of 0.53 (range, 0.42-0.74). The method was also compared with alternative independent analytical techniques for several of the collaborative study samples.
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PMID:Determination of choline in milk and infant formulas by enzymatic analysis: collaborative study. 1069 14

A three-enzyme layered assembly on Au electrodes or Au-quartz crystals, consisting of horseradish peroxidase, HRP, choline oxidase, ChO, and acetylcholine esterase, AChE, is used to sense acetylcholine by the HRP-mediated oxidation of 3,3',5,5'-tetramethylbenzidine, TMB (1), by H2O2, and the formation of the insoluble product (2) on the respective transducers. The analyte-substrate, acetylcholine, is hydrolyzed by AChE to choline that is oxidized by ChO and O2 to yield the respective betaine and H2O2. The amounts of generated H2O2 and the resulting insoluble product on the transducers correlate with the concentration of acetylcholine in the samples. The formation of the insoluble product (2) on electrode supports is followed by faradaic impedance spectroscopy that probes the increased interfacial electron-transfer resistance upon the formation of 2, and by cyclic voltammetry that reflects electron-transfer barriers upon the formation of the precipitate. The frequency of the Au-quartz crystal decreases as a result of the accumulation of the insoluble precipitate. The amount of insoluble product formed on the transducers is controlled by the concentration of acetylcholine and by the time interval of biocatalyzed precipitation. The generation of the insoluble product provides a means to amplify the sensing processes. Acetylcholine concentrations corresponding to 1 x 10(-5) M are easily sensed by the different transducers.
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PMID:Sensing of acetylcholine by a tricomponent-enzyme layered electrode using faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance transduction methods. 1073 94

A novel sensing layer design is presented based on the non-covalent immobilisation of enzymes on derivatized Sepharose beads subsequently entrapped in PVA-SbQ photopolymer. Two different modified Sepharose beads were used, IDA- and DEAE-Sepharose, for the immobilisation, respectively, of horseradish peroxidase (HRP) modified with histidine, and choline oxidase (Chx). The HRP-IDA-Sepharose-based sensing layer was used in a flow injection analysis chemiluminescent system as the basis of an H2O2 biosensor. It was shown that the pre-immobilisation on IDA-Sepharose beads enhanced the sensing layer stability and enabled the immobilisation of a larger amount of enzyme. A 1.8 mg charge of HRP-IDA-Sepharose beads in the sensing layer produced the most sensitive H2O2 biosensor. Such an analytical system exhibited very good performances, with a cycle time of 2 min and a detection limit of 15 pmol (detection ranging over four decades at least), and an unusual long operational stability of 200 measurements (CV, 3.5%). The HRP-IDA-Sepharose beads were then combined with Chx-DEAE-Sepharose. With this modified Sepharose-based biosensor the limit of detection for choline (S/N, 3) was equal to 0.5 pmol and the working range was 0.35 pmol-10 nmol. Moreover, the cycle time was only 2.5 min with the new sensing layer, and a long operational stability of 150 successive assays was found, with a variation coefficient of 2.6%.
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PMID:Chemiluminescent choline biosensor using histidine-modified peroxidase immobilised on metal-chelate substituted beads and choline oxidase immobilised on anion-exchanger beads co-entrapped in a photocrosslinkable polymer. 1128 29

Sphingomyelin is an important lipid component of cell membranes and lipoproteins that can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase and the resultant accumulation of sphingomyelin in cells, tissues, and fluids. In this paper we report a new, enzymatic method to quantify the levels of sphingomyelin in plasma, urine, or tissues from NPD patients and mice. In this assay, bacterial sphingomyelinase is first used to hydrolyze sphingomyelin to phosphorylcholine and ceramide. Alkaline phosphatase then generates choline from the phosphorylcholine, and the newly formed choline is then used to generate hydrogen peroxide in a reaction catalyzed by choline oxidase. Finally, with peroxidase as a catalyst, hydrogen peroxide reacts with the Amplex Red reagent to generate a highly fluorescent product, resorufin. These enzymatic reactions are carried out simultaneously in a single 100-microl reaction mixture for 20 min. Use of a 96-well microtiter plate permits automated and sensitive quantification using a plate reader and fluorescence detector. This procedure allowed quantification of sphingomyelin over a broad range from 0.02 to 10 nmol, similar in sensitivity to a recently described radioactive method using diacylglycerol kinase and 50 times more sensitive than a colorimetric, aminoantipyrine/phenol-based assay. To validate this new assay method, we quantified sphingomyelin in plasma, urine, and tissues from normal individuals and from NPD mice and patients. The sphingomyelin content in adult homozygous or heterozygous NPD mouse plasma and urine was significantly elevated compared to that of normal mice. Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 15 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several Type B NPD patients also was significantly elevated compared to normal individuals of the same age. Based on these results, we propose that this new, fluorescence-based procedure can provide simple, fast, sensitive, and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could also be a useful tool for the study of other sphingomyelin-related diseases and in a variety of research settings where sphingomyelin quantification is required.
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PMID:A fluorescence-based, high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism. 1206 22

A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).
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PMID:Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor. 1248 64


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