毛細管區帶電泳作為快速監測嘌呤5’-核苷酸酶活性的工具：在肌核苷單磷酸及其代謝物之同步偵測分析

Abstract

The purine 5’-nucleotidases (p5’-NT) are involved in the regulation of various physiologically active purine nucleosides, nucleotides, and their analogues in organs. Therefore p5’-NT maintains balanced purine nucleotide pools, so it is one of the important phosphohydrolases. The purpose of the research was to develope a capillary zone electrophoretic method to simultaneously analyze inosine 5’-monophosphate (IMP) and its metabolites, namely inosine (I), hypoxanthine (hXan), xanthine (Xan), and Uric acid. The sample buffer is the p5’-NT assay solution, which contains 50 mM Tris-HCl , 20 mM MgCl2, and 2 mM β-glycerol phosphate at pH 8.0. Addition of acetonitrile and sodium chloride could enhance sample stacking online. In the optimal condition, the four standards, I, hXan, Xan, and Uric acid, were effectively separated in 8 minutes. The method was linear in the range of 5-300 ?嵱 for I and hXan, and 10-400 ?嵱 for Xan and uric acid with the correlation coefficients 0.9999, 0.9999, 0.9997, and 0.9999 for I, hXan, Xan, and Uric acid, respectively. The concentration limits of detection of I, hXan, Xan, and Uric acid were 1.4 ?嵱, 2.1 ?嵱, 5.4 ?嵱, and 3.1 ?嵱, respectively. In the precision test, all relative standard deviations of migration time and area ratio were less than 0.99 % and 5.48 %, respectively. The method was applied to analyze the p5’-NT in the cellular extract of Hep G2. It suggested that p5’-NT from Hep G2 followed Michaelis Menten kinetics . The maximum catalytic velocity and Michaelis constant of Hep G2 p5’-NT were 11.75 nmol/min/mg and 5.97 mM, respectively.