Quantitative Determination of DNA-Binding Parameters for the Human Estrogen Receptor in a Solid-Phase, Nonseparation Assay

Abstract

Binding of transcription factors to specific sequences of DNA has been studied for more than a decade and has become a very productive field of research. This paper describes the application of the recently developed technique of scintillating microtitration plates in the study of protein–DNA interactions. A DNA sequence containing the classical consensus estrogen response element derived from the 5′ upstream regulatory region of the frog vitellogenin gene was immobilized to scintillating microtitration plates. Equilibrium binding studies and kinetic studies were performed with [3H]estradiol labeled human estrogen receptor. The observed equilibrium dissociation constant (Kd) was 2.0 ± 0.3 nMand the observed Hill coefficient of 2.0 indicated a positive cooperativity. Two association rate constants were observed, one slower of 0.3 × 106M−1min−1for lower concentrations of estrogen receptor and one faster of 6.3 × 106M−1min−1for higher concentrations. The dissociation rate was 0.005 min−1. The technique described has a potential in basic research concerning characterization of DNA binding. It is also well suited to applied research as a tool in high-throughput screening of compound libraries in the search of agents inhibiting transcription factor binding to DNA.