反相高效液相色谱法优化嘌呤核苷磷酸化酶检测方法

Optimized assay for purine nucleoside phosphorylase by reversed-phase high-performance liquid chromatography.

作者信息

Halfpenny A P, Brown P R

出版信息

J Chromatogr. 1980 Oct 31;199:275-82. doi: 10.1016/s0021-9673(01)91379-2.

Abstract

The reversed-phase mode of high-performance liquid chromatography (HPLC) was used to assay purine nucleoside phosphorylase (PNPase E.C. 2.4.2.1) in human erythrocytes. The reaction conditions were optimized with respect to pH, concentration of enzyme, concentration of substrate and time. In this method, a sample of erythrocytes was incubated with substrate and necessary cofactors. After termination of the reaction, both the decrease in substrate and the increase in product were measured. HPLC is highly suitable for PNPase as both the forward and reverse reactions can be monitored. The complete separation of products from reactants allows the determination of any competing or side reactions.

摘要

采用高效液相色谱（HPLC）的反相模式测定人红细胞中的嘌呤核苷磷酸化酶（PNPase，酶编号E.C. 2.4.2.1）。针对pH、酶浓度、底物浓度和时间对反应条件进行了优化。在该方法中，将红细胞样品与底物及必要的辅因子一起孵育。反应终止后，测定底物的减少量和产物的增加量。HPLC非常适合用于PNPase，因为正向和反向反应均可监测。产物与反应物的完全分离使得能够确定任何竞争性反应或副反应。