Mechanistic insights into Zika virus NS3 helicase inhibition by Epigallocatechin-3-gallate: Supplementary Files

Deepak Kumar; Nitin Sharma; Murali Aarthy; Sanjeev Singh; Rajanish Giri

The NTPase activity of recombinant NS3 helicase was analyzed by using malachite green method as described previously in literature (45, 46) . We have little modified the ratio of reagents as; 1mg/ml Malachite green, 2mg/ml Ammonium molybdate, 0.7 M HCl and 0.05 % Triton X-100. All the reagents were prepared in ultrapure water ensuring that there is no phosphate contamination. A blank sample O.D. below 0.3 at 630 nm (TECAN infiniteM200PRO) confirms the phosphate free assay system. A phosphate standard curve (serially diluted 6.25 µM -100 µM) was prepared (40 µL sample + 160 µL Malachite reagent) which is used further to quantitate the amount of free phosphate released by NS3 helicase. In 96 well micro-plate assay, NS3 helicase was preincubated in duplicates at a concentration of 80 nM in 20 µl assay buffer (40 mM Tris, 80 mM NaCl, 8 mM Mg(AcO)2, 1 mM EDTA, pH 7.5) . The NTP hydrolysis reaction was started after adding 10 µL ATP in varying concentrations from 25 µM to 2500 µM. Final sample volume was kept 40 µL and after 20 minutes of incubation at 25 0 C, reaction was terminated by adding 160µL of malachite reagent. After incubating the reaction at room temperature for 5 minutes absorbance was measured at 630 nm. All the kinetic parameters were calculated by plotting the data in GraphPad Prims software 7.0. The data fitting was done using Michaelis-Menten equation (V= V max [S]/ (K m + [S]) to calculate kinetic parameters (V max , K m and K cat ).