Figure 6. Competitive binding study of BdorCSP2 and Rhodojaponin-III.

A, CD spectra of BdorCSP2. Spectra were recorded in 50 mM Na phosphate, pH 7.4, at room temperature between 190 and 260 nm. Datum are average value of three independent measurements. B, The binding curve of 1-NPN and relative Scatchard plot analysis (inset). To measure the affinity of 1-NPN to BdorCSP2, the fluorescence of 2 μM 1-NPN in 50 mM Tris-HCl was excited at 337 nm and emission spectra were recorded between 350 nm and 550 nm. And then, 2 μM of protein was added and titrated with aliquots of 1 mM 1-NPN to final concentrations of 2 to 16 μM. The experiment was replicated for at least three times, and the data were analyzed using Prism software and indicated the presence of a single binding site. The dissociation constant was 2.707 μM (±0.11 SEM). C, Competitive binding of the Rhodojaponin-III with BdorCSP2. Final solutions of 2 μM Bdor-CSP2 protein and 2 μM 1-NPN were titrated with Rhodojaponin-III in methanol to final concentrations of 0-200 μM. Datum arean average values of three independent measurements and error bars represented the standard deviations of the mean derived from the differences between the measurements.