Isothermal titration calorimetry to determine association constants for high-affinity ligands
Isothermal titration calorimetry to determine association constants for high-affinity ligands
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DOI：
10.1038/nprot.2006.28
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Freire, Ernesto
中科院分区：
文献类型：
--
作者：
Velazquez-Campoy, Adrian;Freire, Ernesto
药物开发中的一个重要目标是设计对其靶分子具有高结合亲和力的抑制剂和配体。在优化这些相互作用时，一旦结合亲和力接近并超过纳摩尔水平，则精确测定结合亲和力变得越来越困难。等温滴定量热法（ITC）可用于通过使用称为置换滴定的实验模式来确定低至皮摩尔范围的配体的完整结合热力学。在置换滴定中，通过将蛋白质与较弱的竞争性配体预混合，将不能直接测量的高亲和力配体的缔合常数人工降低至可测量的水平。为了执行该方案，必须进行两次滴定：弱配体对靶大分子的直接滴定和高亲和力配体对弱配体-靶大分子复合物的置换滴定。该方案大约需要5小时。
An important goal in drug development is to engineer inhibitors and ligands that have high binding affinities for their target molecules. In optimizing these interactions, the precise determination of the binding affinity becomes progressively difficult once it approaches and surpasses the nanomolar level. Isothermal titration calorimetry ( ITC) can be used to determine the complete binding thermodynamics of a ligand down to the picomolar range by using an experimental mode called displacement titration. In a displacement titration, the association constant of a high- affinity ligand that cannot be measured directly is artificially lowered to a measurable level by premixing the protein with a weaker competitive ligand. To perform this protocol, two titrations must be carried out: a direct titration of the weak ligand to the target macromolecule and a displacement titration of the high- affinity ligand to the weak ligand - target macromolecule complex. This protocol takes approximately 5 h.