An Optimized Assay for Adenosine Deaminase Using Reverse Phase High Pressure Liquid Chromatography

Abstract

A sensitive, optimized assay for adenosine deaminase (E.C. 3.5.4.4), is presented which is based on a reverse phase HPLC analysis of adenosine. In this method a sample of erythrocytes was incubated with adenosine and the decrease in the adenosine concentration with time was analyzed by HPLC. The precision of the method averaged approximately 5% RSD with a sensitivity of about 0.1 U/ml of packed erythrocytes. Comparison with other literature values showed similar activities for adenosine deaminase in erythrocytes (0.229 ± 0.025) U/ml (α = 0.05). The optimization included studies on the ionic strength, pH, enzyme and substrate concentration, and reaction time. The Km for adenosine deaminase was found to be (0.178 ± 0.018) mM (α = 0.05). The method offers several advantages over other assay methods, including an improved capability to discern competing side reactions from other enzymes.