This article is part of the Special Issue dedicated to the memory of Elsa Abuin.
Evaluation of the Number of Binding Sites in Proteins from their Intrinsic Fluorescence: Limitations and Pitfalls†
Article first published online: 26 AUG 2013
© 2013 The American Society of Photobiology
Photochemistry and Photobiology
Volume 89, Issue 6, pages 1413–1416, November/December 2013
How to Cite
Lissi, E., Calderón, C. and Campos, A. (2013), Evaluation of the Number of Binding Sites in Proteins from their Intrinsic Fluorescence: Limitations and Pitfalls. Photochemistry and Photobiology, 89: 1413–1416. doi: 10.1111/php.12112
- Issue published online: 4 NOV 2013
- Article first published online: 26 AUG 2013
- Accepted manuscript online: 21 JUN 2013 10:39AM EST
- Manuscript Accepted: 3 JUN 2013
- Manuscript Received: 23 JAN 2013
- DICYT (USACH)
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 ⇆ QnP. 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.