IC₅₀ determination of compound C2 for PaAhpC.

(A) Aliquots of PaAhpC enzyme were reduced with 5 mM DTT for 1 hour and then desalted to remove excess of reductant. Aliquots containing 10 μM of the reduced and desalted enzymes were treated varying the concentration of compound C2 (5, 10, 25, 50 and 100 μM for 1 hour at room temperature and subsequently desalted again. Next, 3μM of the treated proteins were added to reactions containing 6 μM EcTrx, 0.9 μM EcTrxR, 150 μM NADPH, 50 mM HEPES (pH 7.4), 100 μM DTPA and 1 mM sodium azide. The reactions were incubated at 37°C for 5 minutes before being initiated by the addition of 500 μM H2O2 and monitored spectrophotometrically at 340 nm, 37°C, for 5 minutes. Reactions without treatment with compound C2 were used as positive controls (green square), and reactions without the addition of AhpC were used as negative controls (black square). (B) The initial rates (v0) of each reaction were calculated, transformed into percentual values, and plotted to calculate the IC50 values of compound C2 for PaAhpC. All experiments were performed at least three times in triplicate.