d-Glucose: Determination with Hexokinase and Glucose-6-phosphate Dehydrogenase

Publisher Summary This chapter discusses an enzymatic method for the determination of D -glucose with hexokinase and glucose-6-phosphate dehydrogenase. The optical method for the determination of glucose-6-phosphate, on which the method discussed in the chapter is based, was developed by O. Warburg and his collaborators. Hexokinase catalyzes the phosphorylation of glucose by adenosine triphosphate (ATP). Glucose-6-phosphate is oxidized in the presence of triphosphopyridine nucleotide (TPN) by glucose-6-phosphate dehydrogenase. For all practical purposes, both the reactions proceed stoichiometrically and quantitatively, although reversibility has been demonstrated under special conditions. The reduced triphosphopyridine nucleotide (TPNH) arising in the second reaction is determined spectrophotometrically at 340 or 366 mμ and serves as a measure of the glucose-6-phosphate formed from the glucose in the first reaction. The preparation need not be crystalline but must be relatively free from compounds that interfere with the glucose determination. Sometimes, the preparations still contain glucose, which has been added as a stabilizing agent during purification. It can be removed from highly purified hexokinase by dialysis. The least interference from contaminating enzymes occurs if the specific activity of the hexokinase preparation used approaches that of the crystalline enzyme.

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