Ascorbate oxidase reduced form

Fig. 11. Schematic drawing of the reduced form of ascorbate oxidase around the trinuclear copper site. The included copper-copper distances are the mean values between both subunits.

In the case of laccase and ascorbate oxidase, the observed ET rates for the reduction of the type-3 coppers (see Table VIII) are lower than the observed turnover number. This can be explained only by the possibility that the enzymes are in a resting form under the experimental conditions. A considerable reorganization energy seems to be necessary to get to the reduced state of the type-3 coppers (release of the bridging OH" and movement of the copper GU2 and GU3). From these data it cannot be decided what the rate-limiting step is in the catalytic cycle, either this intramolecular ET or the reaction of the dioxygen at the trinuclear copper site. 

For ascorbic acid analysis, metaphosphoric acid is very useful in the inactivation of the catalytic effect of ascorbic acid oxidase as well as other catalytic oxidizing agents discussed previously. Foods such as fruits and vegetables also have a tendency to have a larger proportion of dehydroascorbic acid than animal tissues consequently, methods that assay for only the reduced form of ascorbate may provide misleading low values. 

Fig. 9. The EPR spectrum of ascorbate oxidase from Cucumber. Native form (solid) and reduced with ascorbate (dashed). [Taken from Nakamura, Makino and Ogura , Ref. (797)]...

The reaction scheme used in the first commercial electrochemical test strip from MediSense (now Abbott Diabetes Care) is shown later. Electron transfer rates between the reduced form of glucose oxidase and ferricinium ion derivatives are very rapid compared with the unwanted side reaction with oxygen (Cass et al. 1984 Forrow et al. 2002). The Abbott Diabetes Care Precision QID strip includes the l,l -dimethyl-3-(2-amino-l-hydroxyethyl) ferrocene mediator, which has the desirable characteristics of high solubility in water, fast electron-shuttling (bimolecular rate constant of 4.3 X 10 M s ), stability, and pH independence of the redox potential (Heller and Feldman 2008). Electrochemical oxidation of the ferrocene derivative is performed at 0.6 V. Oxidation of interferences, such as ascorbic acid and acetaminophen present in blood, are corrected for by measuring the current at a second electrode on the strip that does not contain glucose oxidase. 

Further evidence of like character in support of the participation of coenzyme I in reactions associated with the reduction of dehydroascorbic acid has been advanced by Waygood (1950). Cell-free extracts of wheat seedlings were found to contain a malic dehydrogenase enzyme, reducing coenzyme I, as well as ascorbic oxidase and peroxidase enzymes. When to such extracts malic acid, coenzyme I, and ascorbic acid were added, together with a fixative for the oxalacetate formed in the reaction, the system absorbed oxygen in excess of that required for the complete oxidation of ascorbic acid. In this system methylene blue could replace ascorbic acid. 

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