A New Method of Activity-Staining for Flavin-Containing Oxidase and Some Properties of the Enzymes Obtained with This Technique

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A new staining method for flavin-containing oxidases in the polyacrylamide gel was investigated. This method is based on the formation of red-colored dye, oxidized o-dianisidine, which is produced by the reaction of peroxidase (EC 1.11.1.7; donor: H2O2 oxidoreductase) coupling with a H2O2-producing oxidase in the presence of appropriate substrate. In comparison with the usual proteinstaining method, good results on the activity bands concerned were obtained from the investigation of glucose oxidase (EC 1.1.3.4; β-D-glucose: O2 oxidoreductase), xanthine oxidase (EC 1.2.3.2; xanthine: O2 oxidoreductase) and D-amino acid oxidase (EC 1.4.3.3; D-amino acid: O2 oxidoreductase (deaminating)), and some findings were made by using this technique:1. Commercial xanthine oxidase from buttermilk was separated into two bands which were both active in the presence of hypoxanthine.2. Crystalline D-amino acid oxidase suspended in 1.8M ammonium sulfate showed distinct two bands.3. Apoenzyme of glucose oxidase could be stained by pretreating with FAD solution prior to staining for the holoenzyme.
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