Abstract
Publisher Summary This chapter describes the enzymic method for the determination of fructose 6-phosphate, xylulose 5-phosphate, and sedoheptulose 7-phosphate. Transketolase catalyzes the cleavage of keto sugars to form an active glycolaldehyde, which is then transferred to the respective substrate. In the presence of ferricyanide, the active glycolaldehyde is oxidized to form glycolic acid. The reaction is irreversible and 2 mol of ferricyanide are reduced per mole of the consumed substrate. The reduction of ferricyanide is accompanied by a decrease in the absorbance at 420 nm. Its consumption in the reaction mixture can be calculated by determining the molar extinction coefficient of ferricyanide. In the procedure described in the chapter, two spectrophotometric cuvettes, the experimental and reference, are filled with the following components: 0.05 M glycylglycine buffer, 2 mM MgCl 2 , 0.1 m M thiamine pyrophosphate, and 1.4 m M K 3 Fe (CN) 6 . The formula to calculate the quantity of fructose 6-phosphate is also provided in the chapter. Quantitative determination of other keto compounds is similar to that of fructose 6-phosphate.
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