Assay of Purine Nucleoside Phosphorylase in Erythrocytes by Flow-Injection Analysis with Fluorescence Detection(Analytical,Chemical)

概要

論文の詳細を見る
A sensitive assay for purine nucleoside phosphorylase (PNP) in human erythrocytes is described. Erythrocyte lysate is incubated with the substrate inosine in the presence of phosphate for the enzyme reaction, and the resulting mixture is deproteinized with perchloric acid and neutralized. The supernatant containing the reaction product, hypoxanthine, is applied to a flowinjection system in which immobilized enzyme columns of xanthine oxidase, urate oxidase and horseradish peroxidase are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of hypoxanthine is measured fluorimetrically by reaction with 3-(p-hydroxyphenyl)propionic acid in the system. The lower limit of determination (signal-to-noise ratio= 5) for hypoxanthine is 0.3pmol in a 20-μl injection volume, which corresponds to a PNP activity of 0.117 μmol/min/ml erythrocytes. This method permits the assay of PNP in only 10 nl of human erythrocytes in 50 μl of erythrocyte lysate.
社団法人日本薬学会の論文
1987-11-25