3.2.4. PARP-1 and PARP-2 Enzyme Assay

The reaction of autopoly(ADP-ribosyl)ation was carried out as follows: for PARP-1 50 mM tris-HCl, pH 8.0, 20 mM MgCl2, 150 mM NaCl, and 7 mM β-mercaptoethanol, activated DNA 2 OE/mL, 0.3 mM [32P] NAD+ at 37 ◦C. The reaction was initiated by adding PARP-1 to 200 nM and the reaction mixtures were incubated for 1.5 min. For PARP-2: 50 mM tris-HCl, pH 8.0, 3 mM spermin, 150 mM NaCl, and 7 mM β-mercaptoethanol, activated DNA 2 OE/mL, 0.6 mM [32P] NAD+ at 37 ◦C. The reaction was initiated by adding PARP-2 to 800 nM and the reaction mixtures were incubated for 5 min. The reaction was stopped by placing 10 µL aliquots onto Whatman 1 paper filters soaked with 5 % TCA. The filters were washed with 5% TCA for four times and dried in air after the removal of TCA with 90% ethanol. The incorporation of radioactivity into the product was calculated with a Tri-Carb 2800 scintillation counter (Perkin Elmer, Waltham, MA, USA) according to the Cherenkov method or using Typhoon FLA 9500 scanner (GE Healthcare, Chicago, IL, USA). To determine the IC<sup>50</sup> value of inhibitors (concentration of the compound required reducing the enzyme activity by 50%), we studied

the activity of the enzyme at different concentrations of inhibitors. Measurements were done in at least two independent experiments. IC<sup>50</sup> values were calculated using the Origin Pro 8.0 software by nonlinear regression analysis.

The determination of the kinetic parameters of inhibition Km and Vmax was carried out as described [76]. In all experiments, the points on the experimental curves represent the average of a minimum three independent experiments. Standard deviation did not exceed 10%.
