Figure 3.

D-penicillamine inhibits hydrogen sulfide biosynthesis. (A) Experiments showing the dependency of H₂S production from Cys (1 mM) by human CSE on the concentration of D-pen (up to 100 mM) in 200 mM HEPES at pH 7.4 and 37 °C. The plot (n = 3 ± SD) was fitted with a four-parameter logistic equation and the relative IC₅₀ value is reported in the text. Inset: Representative experiments showing the inhibition of human CSE activity by D-penicillamine in the H₂S assay. The reaction mixture contained cysteine (1 mM), lead acetate (0.4 mM) and varying concentrations of D-pen (indicated on the plot) in 200 mM HEPES, pH 7.4 at 37 °C. H₂S production was initiated by adding human CSE (40 μg) and recorded at 390 nm (formation of a PbS precipitate). (B) Michaelis–Menten plot analysis of CSE inhibition by D-penicillamine. Inhibition of H₂S synthesis was determined in assay mixtures containing 1–10 mM cysteine, 0.4 mM lead acetate, CSE (40 μg) and varying concentrations of D-pen (closed circle, 0 mM; opened circle, 1 mM; closed downward triangle, 5 mM; opened upward triangle, 10 mM) in 100 mM HEPES at pH 7.4 and 37 °C. The data (n ≥ 2 ± SD) were fitted as described in “Experimental Procedures” to extract the CSE-inhibitor competitive (K_ic) and uncompetitive (K_iu) dissociation constants.