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

嘌呤5’-核苷酸酶 (purine 5’-nucleotidase, p5’-NT) 可以調節各組織器官中具各種生理活性的嘌呤核苷、核苷酸及兩者之類似物的濃度，來維持體內核苷酸代謝的平衡，為重要的磷酸酯水解酶。本研究之目的在於開發一毛細管區帶電泳方法，以同步分析肌核苷單磷酸 (Inosine 5’-monophosphate, IMP) 經酵素催化代謝後之相關產物，包括肌苷 (Inosine, I)、次黃嘌 (Hypoxanthine, hXan)、黃嘌呤 (Xanthine, Xan) 與尿酸 (Uric acid)。樣品緩衝液的組成是以p5’-NT之酵素分析緩衝液為主 (50 mM Tris-HCl, 20 mM MgCl2, 2 mM β-glycerol phosphate，pH 8.0)，再另外添加氯化鈉與氰甲烷來造成線上樣品濃縮的效果。在我們所尋找的最佳條件下，可於八分鐘內將四種標準品有效分離，故此分析方法可以用來同步分析IMP及其代謝的相關產物。在此毛細管區帶電泳法中，其線性範圍在5~300和10~400 ?嵱之間，I、hXan、Xan 及 Uric acid 檢量線之相關係數分別為0.9999、0.9999、0.9997 和 0.9999，而偵測極限分別為1.4 ?嵱、2.1 ?嵱、5.4 ?嵱、3.1 ?嵱。於再現性試驗中，I、hXan、Xan 、Uric acid 和內標準品Adenosine 3’,5’-cyclic monophosphate (cAMP) 面積比再現性的相對標準偏差皆小於 5.48 %，滯留時間的相對標準偏差皆小於 0.99 %。初步的真實樣品分析結果顯示：人類肝癌細胞 Hep G2 的p5’-NT最初反應速度與濃度的關係符合 Michaelis Menten 動力學模式，而最大反應速度與Michaelis常數分別為11.75 nmol/min/mg、5.97 mM。

關鍵字

毛細管區帶電泳；肌核苷單磷酸；嘌呤5’-核苷酸酶

並列摘要

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.