NMR spectroscopy is currently extensively used in binding assays for hit identification, but its use in dissociation constant determination is more limited when compared to other biophysical techniques, in particular for tight binders. Although NMR is quite suitable for measuring the binding strength of weak to medium affinity ligands with dissociation constant K_D > 1 μM, it has some limitations in the determination of the binding strength of tight binders (K_D < 1 μM). A theoretical analysis of the binding affinity determination of strong ligands using different types of NMR experiments is provided and practical guidelines are given for overcoming the limitations and for the proper set-up of the experiments. Some approaches require reagents with unique properties or highly specialized equipment, while others can be applied quite generally. We describe all approaches in detail, but give higher emphasis to the more general methods, like competition experiments, where we include actual experimental data and discuss the practical aspects.

核磁共振波谱目前广泛用于命中鉴定的结合测定，但与其他生物物理技术相比，其在解离常数测定中的应用受到更多限制，特别是对于紧密结合物。虽然 NMR 非常适合测量解离常数 K _D > 1 μM 的弱至中等亲和力配体的结合强度，但它在测定紧密结合物 (K _D < 1 μM) 的结合强度方面存在一些局限性。提供了使用不同类型的 NMR 实验对强配体的结合亲和力测定的理论分析，并给出了克服局限性和正确设置实验的实用指南。有些方法需要具有独特性能的试剂或高度专业化的设备，而另一些方法则可以相当普遍地应用。我们详细描述了所有方法，但更加强调更通用的方法，例如竞争实验，其中我们包括实际的实验数据并讨论实际方面。