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

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.