Carlsson B, Häggblad J
Karo Bio AB, Novum, Huddinge, Sweden.
Anal Biochem. 1995 Dec 10;232(2):172-9. doi: 10.1006/abio.1995.0004.
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 nM and the observed Hill coefficient of 2.0 indicated a positive cooperativity. Two association rate constants were observed, one slower of 0.3 x 10(6) M-1 min-1 for lower concentrations of estrogen receptor and one faster of 6.3 x 10(6) M-1 min-1 for 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.
转录因子与特定DNA序列的结合已被研究了十多年，并且已成为一个成果丰硕的研究领域。本文描述了闪烁微孔板这一最新开发的技术在蛋白质-DNA相互作用研究中的应用。将一段含有源自青蛙卵黄蛋白原基因5'上游调控区的经典共有雌激素反应元件的DNA序列固定到闪烁微孔板上。使用[3H]雌二醇标记的人雌激素受体进行平衡结合研究和动力学研究。观察到的平衡解离常数（Kd）为2.0±0.3 nM，观察到的希尔系数为2.0，表明存在正协同性。观察到两个缔合速率常数，较低浓度雌激素受体时较慢的为0.3×10(6) M-1 min-1，较高浓度时较快的为6.3×10(6) M-1 min-1。解离速率为0.005 min-1。所描述的技术在关于DNA结合特性的基础研究中具有潜力。它也非常适合作为一种工具应用于高通量筛选化合物库，以寻找抑制转录因子与DNA结合的试剂的应用研究。
