Ascorbate Oxidase: A New Method of Purification. Characterization of the Purified Enzyme<sup>*</sup>

Tokuyama, Keiko; Clark, Eloise E.; Dawson, Charles R.

doi:10.1021/bi00883a021

Cited by 27 publications

(7 citation statements)

References 11 publications

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“…The preparations exhibit a specific activity in the range of 4025 f 50 units/mg protein, and contain 0.46-0.52 copper corresponding to 10-12 Cui 140000 MI. This metal content is significantly higher than the value of 8 found earlier by Tokoyama et al [7], and it is almost twice as much as that determined by Mondovi et al [8] in most of their preparations.…”

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confidence: 59%

Ascorbate Oxidase from Cucurbita pepo medullosa

Marchesini¹,

Kroneck²

1979

European Journal of Biochemistry

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1. Ascorbate oxidase has been isolated from the green squash Cucurbitapepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20 000 M, could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 f 0.11140000 MI) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 2. The pure enzyme is characterized by the following optical purity indices: A2so/Ablo = 25 +_ 0.5, A330/A610 = 0.65 & 0.05 and A6I0/A500 = 7.0 & 0.25. The molar absorption coefficient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts to 9700 M-' em-'.3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, g , = 2.058, g, = 2.036; A , = 5.0 mT, A , = A , = 0.5 mT, for the type-2 (non-blue) copper gli = 2.242, g l = 2.053; All = 19.0 mT, A1 = 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra.4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductate must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (%ax = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (l/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm ( A s = 1000 f 50 M-'

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confidence: 59%

Ascorbate Oxidase from Cucurbita pepo medullosa

Marchesini¹,

Kroneck²

1979

European Journal of Biochemistry

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“…The pH range (pH 5.6 -7.0) for the catalytic action of ascorbate oxidase 31,32 is very close to that (pH 5.5 -10.5) of GluOx, 29 and hence both enzymes are active at pH 7.0. When the capillary electrode was used without ascorbate oxidase, the oxidation of ascorbate (Fig.…”

Section: Selectivitymentioning

confidence: 76%

“…The interference from ascorbic acid, one of the major components in the brain, was removed by adding ascorbate oxidase to the inner solution. The enzyme catalyzes the oxidation of L-ascorbate to 2-dehydroascorbate 31,32 according to…”

Section: Selectivitymentioning

confidence: 99%

A Glass Capillary Microelectrode Based on Capillarity and Its Application to the Detection of l-Glutamate Release from Mouse Brain Slices

et al. 2003

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The detection of local and dynamic concentration changes of various bioactive substances in the mammalian central nervous system (CNS) is important for understanding the physiological function of excitatory synaptic transmission and metabolism. Many methods have been developed for the in vivo and in vitro detection of bioactive substances in biological fluids, including in vivo voltammetry, 1-3 patch-clamp biosensors, 4-7 microdialysis (MD) combined with various analytical methods 8 and ionselective microelectrode methods. 9 These methods have been widely used for detecting neurotransmitters, second messengers and other bioactive substances that play a key role in neuronal signal transmission.L-Glutamate is an excitatory neurotransmitter, and its release is thought to play a crucial role in brain development, synaptic plasticity, memory formation and neurotoxicity. 10 25 which is much larger than the electrodes for direct measurements, and the time resolution is on the order of minutes. In addition, the recovery of MD sampling is usually low because released L-glutamate is diluted by a perfusion solution. Niwa et al. used a glass capillary, instead of an MD probe, for sampling L-glutamate in a suction mode, followed by flow-through detection at a GluOx/Os-gel-HRP modified electrode. 26 On-line approaches using liquid chromatography, flow injection analysis and capillary electrophoresis in combination with MD probes have also been reported. 8 Improvements of the spatial dissolution of L-glutamate measurements by miniaturizing the size of sampling probe and/or that of enzyme-based electrodes are important, because the neuronal activity that releases and metabolizes L-glutamate is a very fast event, 10 and hence the concentration of Lglutamate detected at a sensing probe depends on the location and distance of the probe from the neurons. For the purpose of miniaturizing the size of the sensing probes, pulled glass capillaries 27,28 have a unique feature: the tip diameters range from submicro-to several µm, and the glass surface has an affinity to lipids and biocompatibility.In the present paper, we describe a new glass capillary microelectrode (tip diameter, ∼12 µm) based on capillarity for measuring L-glutamate. The inner solution provides a reservoir for enzymes, i.e., L-glutamate oxidase and L-ascorbate oxidase, and a built-in three-electrode system Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Japan A new glass capillary microelectrode for L-glutamate is described using pulled glass capillaries (tip size, ∼12.5 µm) with a very small volume (∼2 µl) of inner solution containing glutamate oxidase (GluOx) and ascorbate oxidase. The operation of the electrode is based on capillary action that samples L-glutamate into the inner solution. The enzyme reaction by GluOx generates hydrogen peroxide that is detected at an Os-gel-HRP polymer modified Pt electrode in a three-electrode configuration. The amperometric response behavior of the electrode was characterized in terms of the capillarity, re...

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“…The interference from ascorbic acid, one of the major components in the brain, is removed by adding ascorbate oxidase to the inner solution. The enzyme catalyzes the oxidation of L-ascorbate to 2-dehydroascorbate (Tokuyama et al, 1965;Nakamura et al, 1968) according to…”

Section: Preparation Of a Capillary Sensormentioning

confidence: 99%

A Glass Capillary-Based Microsensor for L-Glutamate in in Vitro Uses

Sugawara¹,

Shoji²

2011

Microsensors

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Ascorbate Oxidase: A New Method of Purification. Characterization of the Purified Enzyme^*

Abstract: Ascorbate oxidase, prepared by a procedure which employs DEAE-cellulose chromatography and starch-column electrophoresis, has been obtained in high purity and in relatively high yield from either yellow or green summer squash. The enzyme had a specific

Cited by 27 publications

References 11 publications

Ascorbate Oxidase from Cucurbita pepo medullosa

Ascorbate Oxidase from Cucurbita pepo medullosa

A Glass Capillary Microelectrode Based on Capillarity and Its Application to the Detection of l-Glutamate Release from Mouse Brain Slices

A Glass Capillary-Based Microsensor for L-Glutamate in in Vitro Uses

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Ascorbate Oxidase: A New Method of Purification. Characterization of the Purified Enzyme*

Abstract: Ascorbate oxidase, prepared by a procedure which employs DEAE-cellulose chromatography and starch-column electrophoresis, has been obtained in high purity and in relatively high yield from either yellow or green summer squash. The enzyme had a specific

Cited by 27 publications

References 11 publications

Ascorbate Oxidase from Cucurbita pepo medullosa

Ascorbate Oxidase from Cucurbita pepo medullosa

A Glass Capillary Microelectrode Based on Capillarity and Its Application to the Detection of l-Glutamate Release from Mouse Brain Slices

A Glass Capillary-Based Microsensor for L-Glutamate in in Vitro Uses

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Ascorbate Oxidase: A New Method of Purification. Characterization of the Purified Enzyme^*