Ascertaining the activity and inhibition of adenosine deaminase via fluorescence-based assays

A fluorescence-based assay for adenosine deaminase (ADA) activity and inhibition, which may also be formatted as an inhibitor discovery assay, is described. It relies on differences in fluorescence between an isothiazolo-based adenosine analogs (^tzA) and its deaminated product, the corresponding inosine derivative (^tzI), which facilitates a real-time monitoring of enzymatic activity. Inhibitors are added to the enzyme-substrate reaction mixture at various concentrations and the fluorescence signal is recorded over 10min. The percent inhibition is calculated from the signal change at 10min relative to the uninhibited reaction. The percent inhibition is plotted against inhibitor concentration and fitted to a Hill curve. IC₅₀ values are then calculated.