ADP-ribose-pNP is a colorimetric substrate for assessing activity of poly(ADP-ribose)polymerase (PARP) enzymes. The absorbance of released p-nitrophenol is determined at 405 nm, and the slope of the calibration curve is used to convert the absorbencies to moles of product generated. With ADP-ribose-pNP as the colorimetric substrate, PARP-1 was determined to have the largest Km and Vmax values (151 uM and 1.30 nmolmin-1mg-1 respectively) followed by tankyrase-1 (82 uM and 18 pmolmin-1mg-1 respectively) and VPARP (46 uM and 2 pmolmin-1mg-1 respectively). This colorimetric substrate can be used to determine the kinetic parameters for PARP-1, tankyrase-1, and VPARP, and to screen small-molecule inhibitors of PARP-1, tankyrase-1, and VPARP. ADP-ribose-pNP-based continuous assay has considerable advantages over standard discontinuous PARP assays, enabling the high throughput screening of PARP-1, tankyrase-1, and VPARP activities and their inhibitors.
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