Measuring binding kinetics of ligands with tethered receptors by fluorescence polarization and total internal reflection fluorescence

Binding kinetics of nuclear receptors and their specific ligands was measured using polarization anisotropy complemented with total internal reflection fluorescence. Binding affinities of tethered full length human estrogen receptor alpha (ERα) with 17β-estradiol, diethylstilbestrol, raloxifene, 4-hydroxytamoxifen, tamoxifen, and genistein were measured to be 100 (as reference), 100, 35, 21, 8, and 1.5, respectively. They agreed with published results. For the first time, rate constants were measured, and off rates were 1.5, 1.5, 1.3, 1.6, 1.7, and 2.3 × 10^-3 s ^-1 while on rates were 11, 10, 3.3, 2.4, 1.0, and 0.26 × 10⁵ M^-1s^-1, respectively. For the antiestrogen drugs, their comparable off-rates correlated well with their equally similar potency. Eleven ginsenosides were screened as potential ligands. None were found to bind to ERα, but Rb1(S) and 20(S)-Rg3 were shown to bind to peroxisome proliferator-activated receptor gamma. The latter finding corroborated strongly with the therapeutic effects of ginsenosides on diabetic mice observed in a separate study. Our method would complement surface plasmon resonance assay for small ligands in the mass range of tens to hundreds of Daltons.