Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, 10, Marie Curie, Ottawa, Ontario, Canada K1N 6N5.
J Photochem Photobiol B. 2012 Jan 5;106:1-17. doi: 10.1016/j.jphotobiol.2011.11.002. Epub 2011 Nov 15.
Steady state fluorescence measurements, due to their relative simplicity and fast and easy implementation, are one of the most employed techniques for evaluating the non-covalent binding of small molecules to proteins. In the present review we discuss the main characteristics of solute binding and the experimental procedures that can be employed for evaluating both, the efficiency of the process and the number of binding sites. It is also discussed the possibility of determining the distance between endogenous fluorophores and non-covalently bound solutes. Albumins (human serum albumin and bovine serum albumin) are considered as model proteins due to their relevance as solute carriers, the extensive available data comprising binding of a large number of solutes, and the reduced number of intrinsic fluorophores which simplifies the data treatment. It is shown that, in spite of the apparent simplicity of the systems, extreme care must be exercised in data treatment and interpretation to avoid misleading results. This applies to the evaluation of binding constants, number of binding sites, and average distance between intrinsic fluorophores and non-covalently bound solutes associated to the proteins.
稳态荧光测量法由于其相对简单、快速和易于实施，是评估小分子与蛋白质非共价结合的最常用技术之一。在本综述中，我们讨论了溶质结合的主要特征以及可用于评估该过程效率和结合位点数量的实验程序。还讨论了确定内源性荧光团与非共价结合的溶质之间距离的可能性。白蛋白（人血清白蛋白和牛血清白蛋白）被认为是模型蛋白，因为它们作为溶质载体具有重要性，广泛可用的数据包括大量溶质的结合，以及内在荧光团的数量减少，这简化了数据处理。结果表明，尽管系统表面上很简单，但在数据处理和解释时必须非常小心，以避免产生误导性结果。这适用于结合常数、结合位点数量以及与蛋白质相关的内源性荧光团和非共价结合的溶质之间的平均距离的评估。
