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Fluorescence displacement method for the determination of receptor-ligand binding constants

✍ Scribed by Petr Kuzmič; Marcia L. Moss; James L. Kofron; Daniel H. Rich

Publisher
Elsevier Science
Year
1992
Tongue
English
Weight
473 KB
Volume
205
Category
Article
ISSN
0003-2697

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✦ Synopsis

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.

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