An optimized assay for adenosine deaminase using reverse phase high pressure liquid chromatography.
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 (alpha = 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 (alpha = 0.05). The method offers several advantages over other assay methods, including an improved capability to discern competing side reactions from other enzymes.
D000243:Adenosine Deaminase / Q000032:analysis* / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D009700:Nucleoside Deaminases / Q000032:analysis*
D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase