用高效液相色谱-固定化碱性磷酸酶柱后水解分析三磷酸肌醇

Assay of Inositol Triphosphate by HPLC Using an Immobilized Alkaline Phosphatase-Loaded Post-Column Reactor.

D-myo-肌醇-1,4,5-三磷酸(IP_3)是许多动物细胞内重要的第二信使之一。分离和检测IP_3对研究多种递质或激素的作用机制和生理功能都有重要的意义。由于IP_3是一种环醇碳水化合物,没有生色团、荧光团或电活性基团,因而不能直接用高效液相色谱(HPLC)-紫外、荧光或电化学检测法来分析。然而在一定条件下,IP_3可被碱性磷酸酶水解产生无机磷酸。

The D-myo-inositol-1,4,5-triphosphate (IP3) in standard solution and extract of human ghost cell was assayed.The procedure involved anion-exchange chromatography,on-line enzymatic hydrolysis of the phosphate esters,and detection of the inorganic phosphate formed.In the range of 1 to 20 n mol,the linear relation between the IP3 amounts and the peak areas was good (r=0.9016,n=6),and the limit of detection was about 1 n mol.