One-Minute High-Performance Liquid Chromatography Assay for 5′-Nucleotidase Using a 20-mm Reverse-Phase Column

A general procedure is described for developing HPLC-based assays for nucleotidases by using a 20-mm C-18 reversed-phase column. The method is applied on the determination of pyrimidine 5′-nucleotidase activity in human erythrocytes lysate and plasma. The activity is detected using isocratic conditions which separate the product (nucleoside) from the substrate (nucleotide) in less than 1 min with 100 mM potassium phosphate, pH 6.0, at a 2 ml/min flow rate. Nucleoside products of the enzymatic reactions are directly quantitated from their ultraviolet absorbances. The activity value closely agreed with that determined by measuring the production of inorganic phosphate. The method does not require prior dialysis of the sample and is 50-fold more sensitive than that based on colorimetric measurements of inorganic phosphate. Furthermore, avoiding the use of radioactive materials offers significant advantages with respect to commonly adopted assays and can be conveniently used for the determination of nucleotidase activity from different sources.