Fluorescence competitive binding assay

To measure the affinity of the recombinant proteins to the fluorescent probe N-phenyl-1-naphthylamine (1-NPN), a 2 μM solution of each target protein in 50 mM Tris-HCl buffer, pH 7.4, was titrated with aliquots of 1 mM 1-NPN in methanol to final concentrations of 1–16 μM. The affinity of the proteins to other ligands was measured in competitive binding assays, where a solution of the protein and 1-NPN, both at the concentration of 2 μM, was titrated with 1 mM methanol solution containing a competitor with concentrations in the range of 5–50 μM. Fluorescence spectra were measured on an F-7000 FL Fluorescence Spectrophotometer (Hitachi) in a 10 mm light path quartz cuvette at 25°C. All were excited at 337 nm with emission and excitation slit of both 10 nm. The emission spectra were recorded between 350 and 500 nm. Data analysis and plot binding curves were accomplished in the Prism software, assuming that the target protein had a 100% activity with a stoichiometry of 1:1 protein: ligand at saturation. All measurements were performed in triplicates and mean values and standard errors are calculated. The dissociation constants of the competitors were calculated from the corresponding IC₅₀-values (the concentration of ligand halving the initial fluorescence value of 1-NPN), by the equation: K_D = [IC₅₀]/1 + [1-NPN]/ K_1-NPN, where [1-NPN] is the free concentration of 1-NPN and K_1-NPN is the dissociation constant of the complex Protein/1-NPN.