Yammine Anthony, Gao Jinlong, Kwan Ann H
School of Life and Environmental Science and Sydney Nano, University of Sydney, NSW 2006, Australia.
Faculty of Dentistry, University of Sydney, NSW 2145, Australia.
Bio Protoc. 2019 Jun 5;9(11):e3253. doi: 10.21769/BioProtoc.3253.
Tryptophan fluorescence quenching is a type of fluorescence spectroscopy used for binding assays. The assay relies on the ability to quench the intrinsic fluorescence of tryptophan residues within a protein that results from changes in the local environment polarity experienced by the tryptophan(s) upon the addition of a binding partner or ligand. The quenching can arise from local changes near the interaction site or from binding-induced conformational changes. In cases where the titrant absorbs at or near the excitation or emission wavelengths of tryptophan, significant quenching can occur even without an interaction. This is known as the inner filter effect. This protocol describes how to use tryptophan fluorescence quenching to investigate the binding affinity of a protein for its partner/ligand and how to check and correct for the inner filter effect. As an example, we measured the binding affinity of the haem-binding protein, HusA, from for haem, and showed how we accounted for the inner filter effect.
色氨酸荧光猝灭是一种用于结合测定的荧光光谱法。该测定依赖于猝灭蛋白质中色氨酸残基固有荧光的能力，这种荧光是由于加入结合伴侣或配体后色氨酸所经历的局部环境极性变化而产生的。猝灭可能源于相互作用位点附近的局部变化，也可能源于结合诱导的构象变化。在滴定剂在色氨酸的激发或发射波长处或附近有吸收的情况下，即使没有相互作用也可能发生显著的猝灭。这被称为内滤效应。本方案描述了如何使用色氨酸荧光猝灭来研究蛋白质与其伴侣/配体的结合亲和力，以及如何检查和校正内滤效应。作为一个例子，我们测量了来自[具体来源未给出]的血红素结合蛋白HusA与血红素的结合亲和力，并展示了我们如何考虑内滤效应。
