Nitroreductase activity assay

Enzyme reducing each of the substrates are measured by monitoring the concomitant oxidation of NADPH by NfsA and NADH by NfsB in a fluorescence based assay. The reduction in fluorescence due to the NADH oxidation (excitation 350 nm, emission 460 nm) and NADPH oxidation (excitation 340 nm, emission 485 nm) is recorded using a TECAN Infinite M200Pro spectrophotometer, in half area solid black microtiter plates. This change in fluorescence directly corelates to the reduction of the nitro-compounds in the reaction. The NfsA assays is performed by initiating the reactions with 40 μM NADPH and 10 nM NfsA in 50 mM Tris-HCl (pH 7.4), 5 mM EDTA against 20 μM substrate (5 μl of compounds/substrate dissolved in 100% DMSO) in a total reaction volume of 100 μl at 25 °C. Similarly, the NfsB assay is also performed by initiating the reactions with 40 μM NADH and 70 nM NfsB in 50 mM Tris-HCl (pH 7.4), 5 mM EDTA against 20 μM substrate in a total reaction volume of 100 μl. These are the optimal conditions arrived at by titrating the enzyme between 2.5 nM–100 nM, NADH/NADPH between 10 and 100 µM, DMSO between 0 and 15% for its tolerance and solubility of the compounds/substrate. All reactions are performed in replicates of two or more and the fluorescence measurements are made at 10 second intervals for 5–10 minutes.