Nucleoside oxidase that was purified to an electrophretically homogeneousstate catalyzed the oxidation of hydroquinone only in the presence of nucleosides simultaneously with the oxidation of nucleosides. From the identification of the reaction products and a chemical balance study, we concluded that the oxidation of hydroquinone proceeded by the following scheme: 2 hydroquinone + O2->2p-quinone + 2H2O. The enzyme also catalyzed the oxidation of various phenolic compounds and strongly colored substances were formed from phenolic compoundswith 4-aminoantipyrine in proportion to the amountof nucleosides. As described in earlier communications,1~3) we found a new enzyme that catalyzes the oxidation of nucleosides to nucleoside-5'-carboxylic acid via nucleoside-5'-aldehyde without a requirement for an exogenous co factor. The enzymewas purified from the cell-free extract of Pseudomonas maltophilia LB-86 by a procedure involving column chromatographies on DEAE-Toyopearl and gel filtration on Sephacryl S-200. The final preparation was homogeneous on polyacrylamide gel electrophoresis. The purified enzyme oxidized various nucleosides but not did attack nucleotides, bases, or ribose. During the investigation on this enzyme, we found that the enzyme catalyzes the oxidation of hydroquinone to pquinone. This oxidation reaction was identical with that of laccase (EC 1.10.3.2), but this activity of nucleoside oxidase appeared only in the presence of nucleoside. This paper describes an identification of the reaction product and the stoichiometry of enzyme reaction using hydroquinone as a substrate. The activities against various phenolic compoundsand the coupling reaction of phenolic compounds and 4-aminoantipyrine are also presented.
The cathodic current for the reduction of benzoquinone at a glassy carbon electrode was increased in the presence of nucleoside oxidase and inosine. This was attributed to the regeneration of benzoquinone from hydroquinone by the laccaselike reaction of the enzyme, the rate of which depended on the concentration of inosine in the solution. Accordingly, an enzyme electrode for nucleosides was constructed by immobilizing nucleoside oxidase behind a dialysis membrane on a carbon paste electrode containing p-benzoquinone or hydroquinone. The current response of the electrode to nucleosides was studied, and the results were discussed on the basis of the laccaselike activity of the enzyme. The nucleoside oxidase-modified electrode was tested as an amperometric enzyme electrode for nucleosides and could be used at least for three weeks.k7W WORDS: Biosensors, nucleosides, nucleoside oxidase. A"TR0DUCTIONConsiderable attention has been given to the coupling of electrode reaction with an enzymatic reaction, in which the substrate of the enzyme can be oxidized or reduced electroenzymatically, i.e., by bioelectrocatalysis [ 1-31. Molecules serving as redox mediators are usually used to achieve the electron transfer coupling, and enzyme electrodes relying on this principle of current response, mediated amperometric enzyme electrodes, have been constructed (4, 51. A variety of redox molecules including redox polymers have been studied for the mediated electron transfer coupling based on glucose oxidase and other oxidoreductases [ 1-31. Similar coupling without mediators has also been reported between ordinary electrodes (such as carbon and metal surfaces), and several kinds of oxidoreductases [ 6-91.This article describes a new type of electroenzymatic coupling different from the above two coupling schemes, which rely on the unique properties of the enzyme used: nucleoside oxidase [NOD] from Pseudomom maltophiria LB-86 (10-131. NOD has a molecular weight of 'To whom correspondence should be addressed. 8 1992 VCH Publishers, Inc. 1040-0397/92/$3.50 + .25 891 892 Ikeda et al.
The enzyme contains 1 mol of covalently bound FAD, 2g atoms of nonheme iron, 2 mol of labile sulfides, and 1 mol of heme per mol enzyme protein. The absorption spectrum of nucleoside oxidase had maxima 278 and 390nm, and shoulders at 343 and 450nm. The enzyme catalyzes the oxidation of various nucleosides, and the Kmvalue for inosine was 4.4 x 10"5 M. The enzyme was most active at pH 5-6, and was most stable between pH 5.0-6.0 and at temperatures below 60°C. The activity was strongly inhibited by /V-bromosuccinimide and potassium cyanide.
A simple and rapid colorimetric method for the measurement of the freshness of fish meat was developed using a nucleoside oxidase-catalyzed oxidative coupling reaction. Fish freshness can be expressed by the Kx value according to the following equation1K x(%) = 100(HxR+Hx)/(IMP +HxR+Hx). To measure the Kx value, hypoxanthine (Hx) and inosine-5'-monophosphate (IMP) were converted to inosine (HxR) by nucleoside phosphorylase and alkaline phosphatase, respectively, and then HxR was oxidized by nucleoside oxidase in the presence of Af-ethyl-7V-(2-hydroxy-3-sulfopropyl)-3,5dimethoxyaniline sodium salt (DAOS)and 4-aminoantipyrine. The amount of colored substance formed was proportional to the amountsof these compounds.Oneassay was completedwithin 10minand good comparative results were obtained between the A , value from the proposed method and from the HPLCmethod. A method for measuring HxR, Hx, and IMP ratios was also developed.
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