Flanagan Colleen A
School of Physiology and Medical Research Council Receptor Biology Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Wits Parktown, Johannesburg, South Africa.
Methods Cell Biol. 2016;132:191-215. doi: 10.1016/bs.mcb.2015.11.004. Epub 2016 Feb 10.
Radioligand binding assays provide sensitive and quantitative information about guanine nucleotide protein G protein-coupled receptor (GPCR) expression and affinity for a wide variety of ligands, making them essential for drug structure-activity studies and basic GPCR research. Three basic radioligand binding protocols, saturation, indirect (competition, displacement, or modulation), and kinetic binding assays, are used to assess GPCR expression (Bmax), equilibrium dissociation constants for radioligands (Kd) and nonradioactive ligands (Ki), association and dissociation rates, and to distinguish competitive and allosteric mechanisms of GPCR-ligand interactions. Nonspecific radioligand binding may be mitigated by appropriate choices of reaction conditions. Radioligand depletion (bound radioactivity >10% of total radioligand), which compromises accuracy of Kd and Ki measurements, can be limited by adjusting receptor concentration and appropriate radioligand choice. Accurate Kd and Ki values in saturation and indirect binding assays depend on binding equilibrium. Equilibration time for high-affinity ligands, with slow dissociation rates, may require much extended incubation times or increased incubation temperature.
放射性配体结合测定可提供有关鸟嘌呤核苷酸蛋白G蛋白偶联受体（GPCR）表达以及对多种配体的亲和力的灵敏且定量的信息，使其对于药物构效关系研究和基础GPCR研究至关重要。三种基本的放射性配体结合实验方案，即饱和实验、间接实验（竞争、置换或调节）和动力学结合实验，用于评估GPCR表达（最大结合容量Bmax）、放射性配体的平衡解离常数（Kd）和非放射性配体的抑制常数（Ki）、结合和解离速率，并区分GPCR-配体相互作用的竞争性和变构机制。通过适当选择反应条件可以减轻非特异性放射性配体结合。放射性配体耗竭（结合的放射性>总放射性配体的10%）会影响Kd和Ki测量的准确性，可以通过调整受体浓度和适当选择放射性配体来加以限制。饱和结合实验和间接结合实验中准确的Kd和Ki值取决于结合平衡。对于解离速率较慢的高亲和力配体，平衡时间可能需要延长孵育时间或提高孵育温度。
