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. Author manuscript; available in PMC: 2016 Aug 3.

Published in final edited form as: FEBS J. 2015 Aug 3;282(16):3199–3217. doi: 10.1111/febs.13359

Fig. 4 — Time-dependent inactivation of Schistosoma mansoni thioredoxin glutathione reductase by compounds 7 (panels A and B) or 9 (panel C). Panels A-B: SmTGR (40 nM in 100 μL) was incubated in the presence of 100 μM NADPH and various inhibitor concentrations at 25°C for different times of incubation periods (0, 2.5, 5, and 10 min). Compound 7 concentrations used were 0 (●), 0.1(□), 0.3 (◆), 1.0 (◇), and 3.0 (▼) μM (panel A). The k_obs data versus [I] were fitted to equation 5 (see text) which resulted in the hyperbolic curve (dashed line). At low inhibitor 7 concentration the dissociation constant and the first order rate constant for irreversible inactivation were determined as K_I = 0.8 ± 0.08 μM and k_i as 0.57 ± 0.02 min⁻¹, respectively. Panel C: SmTGR (40 nM in 100 μL) was incubated in the presence of 100 μM NADPH and inhibitor at varying concentrations (0 – 0.03 – 0.1 – 0.3 – 1.0 – 3.0 – 10.0 – 30.0 μM) for 0 (●) and (■) 5 min-incubation periods. Reactions were also carried out in the absence of NADPH with the pre-reacted enzyme for 5 min (□). Then, 5 μL aliquots were removed and the remaining activity was measured in a standard DTNB reduction assay as described under Experimental Procedures. 3.4% final DMSO was present in all incubation mixtures.

Fig. 4 — Time-dependent inactivation of Schistosoma mansoni thioredoxin glutathione reductase by compounds 7 (panels A and B) or 9 (panel C). Panels A-B: SmTGR (40 nM in 100 μL) was incubated in the presence of 100 μM NADPH and various inhibitor concentrations at 25°C for different times of incubation periods (0, 2.5, 5, and 10 min). Compound 7 concentrations used were 0 (●), 0.1(□), 0.3 (◆), 1.0 (◇), and 3.0 (▼) μM (panel A). The k_obs data versus [I] were fitted to equation 5 (see text) which resulted in the hyperbolic curve (dashed line). At low inhibitor 7 concentration the dissociation constant and the first order rate constant for irreversible inactivation were determined as K_I = 0.8 ± 0.08 μM and k_i as 0.57 ± 0.02 min⁻¹, respectively. Panel C: SmTGR (40 nM in 100 μL) was incubated in the presence of 100 μM NADPH and inhibitor at varying concentrations (0 – 0.03 – 0.1 – 0.3 – 1.0 – 3.0 – 10.0 – 30.0 μM) for 0 (●) and (■) 5 min-incubation periods. Reactions were also carried out in the absence of NADPH with the pre-reacted enzyme for 5 min (□). Then, 5 μL aliquots were removed and the remaining activity was measured in a standard DTNB reduction assay as described under Experimental Procedures. 3.4% final DMSO was present in all incubation mixtures.