醛缩酶-DNPH比色法测定D-果糖-1,6-二磷酸盐的含量

Determination of Fructose-1,6-Diphosphate with Aldolase DNPH by the Colorimetric Method

对多酶法测定果糖二磷酸盐含量提出改进 ,仅使用一种具有专属性的醛缩酶。醛缩酶催化果糖 - 1,6 -二磷酸 (FDP)降解 ,生成的磷酸二羟丙酮和 3-磷酸甘油醛直接与 2 ,4-二硝基苯肼缩合成紫红色的苯腙。在 5 40 nm波长处测定吸收度。本法测定 FDP有专属性高、省去三种酶试剂。

This paper presents a modified method of enzymatic assay for Fructose 1,6 diphosphate(FDP). FDP is split to dihydroxyacetone phosphate (DAP) and glyceraldehyde 3 phosphate (GAP) by the action of aldolase. DAP is hydrolyzed at room temperature to free triose. Under alkaline conditions, the free triose is reacted with 2, 4 dinitrophenylhydrazine (DNPH), yielding a 2,4 dinitrophenylhydrazine derivative which dissolve in alkali forming a purple color mixture, with maximum absorption at 540 nm. It is proportional to the contents of FDP. Because the method depends on the colorimetric determination of triose formed from fructose 1,6 diphosphate only by aldolase, glycerophosphate dehydrogenase/triosephosphate isomerase (GDH/TIM) and reduced nicotinamide adenine dinucleotide (NADH) which usually applied in multienzymatic method, are omitted in the modified method. The method is specific, convenient and accuracy for the determination of FDP.