Dihydroxyacetone Phosphate, Fructose-l,6-diphosphate and d-Glyceraldehyde-3-phosphate: Determination with Glycerol-1-Phosphate Dehydrogenase, Aldolase and Triosephosphate Isomerase

摘要: Publisher Summary This chapter elaborates the method for enzymatic determination of dihydroxyacetone phosphate, fructose-1, 6-diphosphate, and D -glyceraldehyde-3-phosphate using glycerol-l-phosphate dehydrogenase, aldolase, triosephosphate isomerase. The same indicator reaction can be used spectrophotometric fructose diphosphate glyceraldehyde phosphate. phosphate is based on principle that dehydrogenase (GDH) catalyzes reduction (DAP) with reduced diphosphopyridine nucleotide (DPNH). equilibrium constant this Kc 1.78 × 1011 (moles/1)−1 at pH 0 25°C; even 7 (Kc’ = 104 (moles/1)−1) lies far to right so reacts quantitatively. With a measurable excess DPNH sufficient enzyme proceeds rapidly. decrease in optical density 340 or 334 (also 366) mμ because oxidation measure reaction. In case fructose-1,6-diphosphate, aldolase cleaves fructose-1,6-diphosphate (FDP) give glyceraldehyde-3-phosphate (GAP) (DAP). GAP isomerized DAP by isomerase (TIM) GDH.