Kuzmic P, Moss M L, Kofron J L, Rich D H
School of Pharmacy, University of Wisconsin-Madison 53706.
Anal Biochem. 1992 Aug 15;205(1):65-9. doi: 10.1016/0003-2697(92)90579-v.
The equilibrium constant for the binding of a spectroscopically invisible ligand to its protein receptor can be determined in a competition experiment, by using a structural analog that contains a reporter group (fluorophor). A novel mathematical treatment of the multiple equilibria allows the analysis to be performed under tight-binding conditions. The equilibrium equation for mixtures of two mutually competitive tight-binding ligands can be expressed in a recursive form, a form in which the dependent variable appears on both sides and the solution is found iteratively. The algorithm is also applicable to the special case of weak binding, where the concentration of the bound ligand can be neglected in the mass balance. The fluorescence displacement method is demonstrated on the determination cyclophilin binding to cyclosporin A (CsA), in competition with its fluorescent derivative, [D-Lys(Dns)]8-CsA.
光谱学上不可见的配体与其蛋白质受体结合的平衡常数，可在竞争实验中通过使用含有报告基团（荧光团）的结构类似物来测定。对多重平衡进行的一种新颖数学处理，使得分析能够在紧密结合条件下进行。两种相互竞争的紧密结合配体混合物的平衡方程，可以用递归形式表示，即因变量出现在方程两边且通过迭代求解。该算法也适用于弱结合的特殊情况，即在质量平衡中可忽略结合配体的浓度。通过荧光位移法测定了亲环蛋白与环孢菌素A（CsA）的结合，实验中其荧光衍生物[D - Lys(Dns)]8 - CsA作为竞争物。
