Abstract
Publisher Summary This chapter discusses the determination of ascorbic acid oxidase. The assay method most generally used depends on the fact that, within a certain range of enzyme concentration, the rate of oxygen consumption during the oxidation of L-ascorbic acid is proportional to the amount of enzyme present. One unit of ascorbic acid oxidase activity is defined as that amount of enzyme that causes an initial rate of oxygen uptake of 10 μl. per minute. Specific activity is expressed as units per milligram of dry weight. The preparation of highly purified ascorbic acid oxidase is attempted from eleven different plants and found the yellow summer squash , Cucurbita pepo condensa , to be the most satisfactory source. Procedures developed have resulted in the preparation of solutions of enzyme that are homogeneous electrophoretically and in the ultracentrifuge. Ascorbic acid oxidase from squash is a blue protein having a molecular weight of 150,000, a copper content of 0.25%, and properties of a globulin. Maxima in its absorption spectra appear at 288 and 605 mμ. Homogeneous ascorbic acid oxidase is shown to possess a specific activity of 2000 units/mg. and 750 units/γ of copper. Purified ascorbic acid oxidase shows marked specificity for L-ascorbic acid. D-Ascorbic acid and a number of other ene-diols similar in structure to ascorbic acid are also oxidized although much more slowly.
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