Evaluation of inhibition properties of compound C2 over HsPrx2 and the bacterial thiol proteins EcTrx and EcTrxR.

The enzymes HsPrx2, EcTrx and EcTrxR were reduced with DTT and desalted, then treated with 50 molar equivalents of compound C2 for one hour at RT (red square). The proteins were desalted again to remove excess compound and their peroxidase activity was analyzed by NADPH oxidation assay. (A) For Prx2, reactions were performed containing 5 μM of Prx2, 10 μM of ScTrx1, 0.3 μM of ScTrxR1, 150 μM of NADPH, 50 mM of HEPES (pH = 7.0), 100 μM of DTPA and 1 mM of sodium were used. For EcTrx (B) and EcTrxR (C) reactions consisted of 3 μM of PaAhpC, 6 μM of EcTrx, 0.9 μM of EcTrxR, 150 μM of NADPH, 50 mM of HEPES (pH = 7.4), 100 μM of DTPA and 1 mM sodium azide were used. Prior to experiments the reactions were incubated at 37°C/5 min and reactions were started by the addition of H2O2 (500 μM) and monitored spectrophotometrically at 340 nm, 37°C, for 5 minutes. The positive controls contained the proteins HsPrx2, EcTrx or EcTrxR without prior treatment with compound C2 (green square) and the negative control without the addition of PaAhpC (EcTrx or EcTrxR) (black square). The enzymes samples previously treated with NEM were used as control of protein inhibition (blue square). The experiments were performed three times using triplicates.