Homogeneous competitive assay of ligand affinities based on quenching fluorescence of tyrosine/tryptophan residues in a protein via Főrster-resonance-energy-transfer
作者:Yanling Xie、Xiaolan Yang、Jun Pu、Yunsheng Zhao、Ying Zhang、Guoming Xie、Jun Zheng、Huidong Yuan、Fei Liao
DOI:10.1016/j.saa.2010.08.021
日期:2010.11
A new homogeneous competitive assay of ligand affinities was proposed based on quenching the fluorescence of tryptophan/tyrosine residues in a protein via Főrster-resonance-energy-transfer using a fluorescent reference ligand as the acceptor. Under excitation around 280 nm, the fluorescence of a protein or a bound acceptor was monitored upon competitive binding against a nonfluorescent candidate ligand
基于荧光参考配体作为受体,通过弗斯特共振能量转移淬灭蛋白质中色氨酸/酪氨酸残基的荧光,提出了一种配体亲和力的新的均质竞争测定法。在280nm附近的激发下,在与非荧光候选配体竞争结合时监测蛋白质或结合的受体的荧光。讨论了用任一荧光信号推导受体结合率的化学计量学;离解常数(K d从其取代受体的50%结合的浓度计算非荧光候选配体的)。N-生物素-N'-(1-萘基)-乙二胺(BNEDA)和N-生物素-N'-丹酰基-乙二胺(BDEDA)被用作链霉亲和素的参考配体和受体,以测试这种新的均相竞争性测定。受体与链霉亲和素结合后,链霉亲和素荧光在340 nm处猝灭,BNEDA在430 nm处或BDEDA在525 nm处具有特征荧光。BNEDA和BDEDA的K d是通过竞争性结合生物素获得的。通过定量BNEDA荧光K d通过使用BNEDA或BDEDA作为受体对链霉亲和素荧光进行定量,每种测试的非荧光生物素衍生