Addition Of Ascorbate Oxidase Research Articles

An enzyme-centric approach for constructing an amperometric L-malate biosensor with a long and programmable linear range.

L-Malate is a key flavour enhancer and acidulant in the food and beverage industry, particularly winemaking. Enzyme-based amperometric biosensors offer convenience for monitoring its concentration. However, only a small number of off-the-shelf malate-oxidising enzymes have been used in previous devices. These typically have linear ranges poorly suited for the L-malate concentrations found in fruit processing and winemaking, making it necessary to use precisely diluted samples. Here we describe a pipeline of database-mining, gene synthesis, recombinant expression and spectrophotometric assays to characterise previously-untested enzymes for their suitability in biosensors. The pipeline yielded a bespoke biocatalyst-the Ascaris suum malic enzyme carrying mutation R181Q [AsME(R181Q)]. Our first prototype with AsME(R181Q) had an ultra-wide linear range of 50-200 mM L-malate, corresponding to concentrations found in undiluted fruit juices (including grape). Changing the dication from Mg2+ to Mn2+ increased sensitivity five-fold and adding citrate (100 mM) increased it another six-fold, albeit decreasing the linear range to 1-10 mM. To our knowledge, this is the first time an L-malate biosensor with a tuneable combination of sensitivity and linear range has been described. The sensor response was also tested in the presence of various molecules abundant in juices and wines, with ascorbate shown to be a potent interferent. Interference was mitigated by the addition of ascorbate oxidase, allowing for differential measurements on an undiluted, untreated wine sample that corresponded well with commercial L-malate testing kits. Overall, this work demonstrates the power of an enzyme-centric approach for designing electrochemical biosensors with improved operational parameters and novel functionality. This article is protected by copyright. All rights reserved.

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

Measurement of Total Antioxidant Capacity by Electrogenerated Iodine at Disposable Screen Printed Electrodes

AbstractTotal antioxidant capacity is an important parameter for the evaluation of the oxidative status in different kinds of biological samples such as plant extracts, or in food industry. We report a fast, easy, portable, cost‐effective electroanalytical method to measure total antioxidant capacity, based on the reaction of natural antioxidants with electrogenerated iodine using disposable platinum screen‐printed electrodes. This reaction can be measured by the increment of the electrochemical current signal of iodide oxidation to iodine during a voltammetric cycle. Iodine reacts with reducing compounds such as glutathione, ascorbate, gallic acid and NADH without interference of the corresponding oxidized counter‐parts. The addition of ascorbate oxidase also allows the concentration of ascorbate to be determined. The method was tested with real samples of plant extracts and the results correlated well with those obtained with a standard spectrophotometric method.

Simultaneous Determination of Reactive Oxygen and Nitrogen Species in Mitochondrial Compartments of Apoptotic HepG2 Cells and PC12 Cells Based On Microchip Electrophoresis–Laser-Induced Fluorescence

Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the first application of a microchip electrophoresis-laser-induced fluorescence (MCE-LIF) method for concurrent determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), i.e., superoxide (O(2)(-•)) and nitric oxide (NO) in mitochondria, was developed using fluorescent probes 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM), respectively. Potential interference of intracellular dehydroascorbic acid (DHA) and ascorbic acid (AA) for NO detection with DAF-FM was eliminated through oxidation of AA with the addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescent products of O(2)(-•) and NO, DBZTC oxide (DBO), and DAF-FM triazole (DAF-FMT) showed excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric field of 500 V/cm. The levels of DBO and DAF-FMT in mitochondria isolated from normal HepG2 cells and PC12 cells were evaluated using this method. Furthermore, the changes of DBO and DAF-FMT levels in mitochondria isolated from apoptotic HepG2 cells and PC12 cells could also be detected. The current approach was proved to be simple, fast, reproducible, and efficient. Measurement of the two species with the method will be beneficial to understand ROS/RNS distinctive functions. In addition, it will provide new insights into the role that both species play in biological systems.

The NADH oxidase activity of the plasma membrane of synaptosomes is a major source of superoxide anion and is inhibited by peroxynitrite.

Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate-dependent NADH oxidase activity of 35-40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10-fold lower than that measured for NADH. Ascorbate-dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 +/- 0.5 nmol AFR/min/(mg protein). NADH-dependent *O2- production by PMV occurs with a rate of 35 +/- 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 micro g/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH-dependent *O2- production are identical. Treatment of PMV with repetitive micromolar ONOO- pulses produced almost complete inhibition of the ascorbate-dependent NADH oxidase and *O2- production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5-fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 microm ONOO- pulses lowers the K0.5 for cytochrome c stimulation from 6 +/- 1 (control) to 1.5 +/- 0.5 microm. Thus, the ascorbate-dependent plasma membrane NADH oxidase activity can act as a source of neuronal.O2-, which is up-regulated by cytosolic cytochrome c and down-regulated under chronic oxidative stress conditions producing ONOO-.

Role of ascorbic acid in transferrin-independent reduction and uptake of iron by U-937 cells

The role of ascorbic acid in transferrin-independent ferric iron reduction and uptake was evaluated in cultured U-937 monocytic cells. Uptake of 55Fe by U-937 cells was doubled by 100 μM extracellular ascorbate, and by pre-incubation of cells with 100 μM dehydroascorbic acid, the two-electron-oxidized form of ascorbate. Reduction of extracellular ferric citrate also was enhanced by loading the cells with dehydroascorbic acid. Dehydroascorbic acid was taken up rapidly by the cells and reduced to ascorbate, such that the latter reached intracellular concentrations as high as 6 mM. However, some ascorbate did escape the cells and could be detected at concentrations of up to 1 μM in the incubation medium. Further, addition of ascorbate oxidase almost reversed the effects of dehydroascorbic acid on both 55Fe uptake and ferric citrate reduction. Thus, it is likely that extracellular ascorbate reduced ferric to ferrous iron, which was then taken up by the cells. This hypothesis also was supported by the finding that during loading with ferric citrate, only extracellular ascorbate increased the pool of intracellular ferrous iron that could be chelated with cell-penetrant ferrous iron chelators. In contrast to its inhibition of ascorbate-dependent ferric iron reduction, ascorbate oxidase was without effect on ascorbate-dependent reduction of extracellular ferricyanide. This indicates that the cells use different mechanisms for reduction of ferric iron and ferricyanide. Therefore, extracellular ascorbate derived from cells can enhance transferrin-independent iron uptake by reducing ferric to ferrous iron, but intracellular ascorbate neither contributes to this reduction nor modifies the redox status of intracellular free iron.

茶中のアスコルビン酸測定に関する研究

A spectrophotometer, O2 electrode, and HPLC were used to measure ascorbic acid in green tea cultivated by hydroponics. In the spectrophotometry, ascorbic acid was analyzed by absorbance changes at 265 nm upon addition of ascorbate oxidase, and using an O2 electrode, ascorbic acid was estimated by O2 uptake upon addition of ascorbate oxidase. The apparatus for HPLC was a Hitachi L-6000 model liquid chromatograph with a UV detector set at 244 nm, and separation was done on a Shimpack-CLC-NH2 column. The accuracy of these three methods gave 98.6%∼104.5% recoveries and 0.2%∼1.76% coefficients of variation. The measurement of ascorbic acid in green tea extracts was done using four methods including hydrazine, and equal values were obtained. Application of these three methods to analysis of ascorbic acid were rapid, simple, and equal to or more sensitive than the hydrazine method. After the treatment of dehydroascorbic acid with dithiothreitol, dehydroascorbic acid was estimated by HPLC. Dehydroascorbic acid was completely reduced with a ten-fold excess of dithiothreitol within l0 min at room temperature. This simple procedure was used in the analysis of dehydroascorbic acid in green tea extracts.

Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions

Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb 561. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb α-band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.

Inhibition of dimeric dihydrodiol dehydrogenases of rabbit and pig lens by ascorbic acid

The dehydrogenase activity of dimeric dihydrodiol dehydrogenases (DD) purified from pig and rabbit lenses was inhibited by either L-ascorbic acid or its epimer, isoascorbic acid, at pH 7.5. Isoascorbate [IC50 (concn. giving 50% inhibition) = 0.043 mM for the pig enzyme; IC50 = 0.13 mM for the rabbit enzyme] was a more potent inhibitor than ascorbate (IC50 values 0.45 and 0.90 mM respectively), but 1 mM-dehydroascorbate gave less than 30% inhibition. Glucose, glucuronate, gulono-gamma-lactone, glutathione and dithiothreitol did not inhibit the enzyme activity. The inhibition by isoascorbate and ascorbate was instantaneous and reversible, and their inhibitory potency was decreased by addition of ascorbate oxidase. In the reverse reaction, isoascorbate and ascorbate gave low IC50 values of 0.013 and 0.10 mM respectively for the pig enzyme and 0.025 and 0.25 mM for the rabbit enzyme. The inhibition patterns by the two compounds were competitive with respect to dihydrodiols of naphthalene and benzene and uncompetitive with respect to NADP+, but those in the reverse reaction were uncompetitive with respect to both carbonyl substrate and NADPH. The steady-state kinetic measurements in the forward and reverse reactions by the pig enzyme were consistent with an ordered Bi Bi mechanism, in which NADP+ binds to the enzyme first and NADPH leaves last. The results indicate that ascorbate and its epimer directly bind to an enzyme: NADP+ binary complex as dead-end inhibitors. Thus ascorbate may be an important modulator of DD in the lens.

Hypothalamic peptidyl-glycine alpha-amidating monooxygenase: preliminary characterization

An enzymatic activity capable of converting mono-[125I]-D-Tyr-Val-Gly into mono-[125I]-D-Tyr-Val-NH2 was identified in a crude mitochondrial/synaptosomal preparation from rat hypothalamus. Further subcellular fractionation studies localized a majority of this enzymatic activity to fractions enriched in synaptic vesicles. The alpha-amidation activity demonstrated optimal activity at pH 7.5 to 8, was stimulated by the presence of copper ions and reduced ascorbate and required the presence of molecular oxygen. Endogenous alpha-amidation activity was inhibited by the addition of ascorbate oxidase. Kinetic analyses demonstrated Michaelis-Menten type kinetics for D-Tyr-Val-Gly as the varied substrate with the values of Km and Vmax increasing as the ascorbate concentration in the reaction increased. A variety of peptides possessing carboxyl-terminal glycine residues were potent inhibitors of the reaction, while peptides lacking a carboxyl-terminal glycine residue were not, suggesting that many glycine-extended peptides may serve as substrates in the alpha-amidation reaction. The characteristics of hypothalamic alpha-amidation activity are similar to those previously reported for the alpha-amidation activity in rat pituitary and mouse corticotropic tumor cells suggesting the presence of closely related enzymes in these tissues.