125] Triphosphopyridine and diphosphopyridine nucleotide oxidases

Abstract

Publisher Summary This chapter discusses the determination of triphosphopyridine and diphosphopyridine nucleotide oxidases; and describes the assay methods for old yellow enzyme and new yellow enzyme. In the case of old yellow enzyme, the assay method is based on the manometric determination of the oxygen uptake in the following system: glucose-6-phosphate-Zwischenferment-TPN-yellow enzyme-molecular oxygen. Under the specified conditions the concentration of TPN is high, its reduction proceeds rapidly, and the concentration of the yellow enzyme is ratedetermining in the consumption of oxygen. Catalase, present as an impurity in the Zwischenferment preparation, is inhibited by cyanide to prevent the decomposition of H 2 O 2 . In the case of new yellow enzyme, G-6-P is oxidized by molecular oxygen, and the oxygen consumption, which is a function of the concentration of the new yellow enzyme, is measured manometrically. Both the leuco forms of the old and the new yellow enzyme are rapidly oxidized by methylene blue, but the new yellow enzyme is reduced four times as fast by TPNH, and it is oxidized seven times as slowly by molecular oxygen as the old yellow enzyme. This difference in activity permits a quantitative distinction of the two enzymes under the test conditions outlined in the chapter.