Abstract
A miniaturized fluorescent assay is described that monitors the conversion of NADPH to NADP(+). The same assay format may also be used to measure NADH to NAD(+) conversion. Examples of assay development and validation results are presented to illustrate the use of this protocol to screen an enzyme that consumes NADPH as a cofactor during conversion of substrate to a reduced product. Enzymatic assays are carried out low volume 384-well plates, in which the turnover of NADPH is monitored by the decrease in fluorescent emission at 460 nm between an initial fluorescence measurement and a second reading after 90 minutes. A follow-up assay is used to rule out false positive artifacts arising from compounds that fluoresce at 460 nm.