Lissi Eduardo, Calderón Cristian, Campos Ana
Faculty of Chemistry and Biology, University of Santiago de Chile, USACH, Santiago, Chile.
Photochem Photobiol. 2013 Nov-Dec;89(6):1413-6. doi: 10.1111/php.12112. Epub 2013 Aug 26.
Changes in the intrinsic protein fluorescence with the additive concentration provide one of the most employed methodologies for the evaluation of the binding constant and the number of binding sites. In the last years, more than 175 studies have been published where the double logarithmic plot shown below is used toward determining the number of equivalent binding sites (n). Log [(F° - F)/F] = log K + n log [Q0 ]. However, the value of n evaluated by this procedure is unrelated to the number of equivalent binding sites; rather it represents the stoichiometry of the binding step. The confusion on the meaning of n arises upon assuming that the binding process is represented by the forward and backward elementary steps shown below, implying that binding of the n solutes takes place simultaneously, i.e. there are no intermediate species. nQ + P ⇆ Qn P. The conclusion that n is unrelated to the number of equivalent binding sites is supported by the fact that in all the systems considered (99% of them) n values are close to one and much smaller than those obtained by ultrafiltration. It is then remarkable, the profusion of publications in peer-reviewed, specialized journals including a conceptual error that confuses Hill's coefficient and/or the stoichiometry of the binding step with the number of independent binding sites. Here, we discuss the origin of this common misconception and provide alternative methods to determine the number of binding sites.
随着添加剂浓度的变化，内在蛋白质荧光的改变提供了一种最常用的评估结合常数和结合位点数量的方法。在过去几年中，已经发表了175多篇研究，其中使用如下所示的双对数图来确定等效结合位点的数量（n）。Log[(F° - F)/F] = log K + n log[Q0]。然而，通过该程序评估的n值与等效结合位点的数量无关；相反，它代表结合步骤的化学计量。对n含义的混淆源于假设结合过程由如下所示的正向和反向基本步骤表示，这意味着n个溶质的结合同时发生，即不存在中间物种。nQ + P ⇆ QnP。n与等效结合位点数量无关这一结论得到以下事实的支持：在所有考虑的系统中（其中99%），n值接近1且远小于通过超滤获得的值。那么，值得注意的是，在同行评审的专业期刊中有大量的出版物存在一个概念性错误，即将希尔系数和/或结合步骤的化学计量与独立结合位点的数量混淆。在此，我们讨论这种常见误解的起源，并提供确定结合位点数量的替代方法。
