Mocz Gabor, Ross Justin A
Pacific Biosciences Research Center, University of Hawaii, Honolulu, HI, USA.
Methods Mol Biol. 2013;1008:169-210. doi: 10.1007/978-1-62703-398-5_7.
Fluorescence spectroscopy may serve as a universal tool for the study of protein-ligand interactions. Applications of fluorometry have made use of various aspects of fluorescence such as intensity, emission and excitation spectra, lifetime, quantum yield, polarization state, and anisotropy, as well as energy transfer and other electronic phenomena. An experimentalist has to consider each of these characteristics carefully, frequently in combination with each other, for the analysis of protein-ligand complexes and for the determination of binding constants. Most of the available techniques are of a rather general nature and a wealth of possibilities exists for their utilization. In this chapter we will provide a short survey of selected techniques that can be used for measuring binding constants and probing protein-ligand interactions. Basic principles and phenomena are discussed followed by experimental considerations and examples of binding constant determination. Emphasis is placed on steady-state techniques that employ the use of intrinsic protein fluorescence, labeled ligands, as well as anisotropy and resonance energy transfer.
荧光光谱法可作为研究蛋白质-配体相互作用的通用工具。荧光测定法的应用利用了荧光的各个方面，如强度、发射光谱和激发光谱、寿命、量子产率、偏振态和各向异性，以及能量转移和其他电子现象。实验人员在分析蛋白质-配体复合物和测定结合常数时，必须仔细考虑这些特性中的每一个，而且常常要将它们结合起来考虑。大多数现有技术具有相当普遍的性质，并且在利用它们方面存在大量可能性。在本章中，我们将简要概述一些可用于测量结合常数和探究蛋白质-配体相互作用的技术。先讨论基本原理和现象，接着是实验注意事项以及结合常数测定的示例。重点放在采用蛋白质固有荧光、标记配体以及各向异性和共振能量转移的稳态技术上。
