Signal and binding. II. Converting physico-chemical responses to macromolecule–ligand interactions into thermodynamic binding isotherms

Evaluating the Number of Ligand Binding Sites on Protein from Tryptophan Fluorescence Quenching under Typical Experimental Conditions

“…In this paper, we focused on the case of tryptophan fluorescence quenching in proteins upon binding and tried to suggest a simple algorithm for FQT data processing for the curves measured at typical experimental conditions. As some of the authors before [14][15], we came to the conclusion that measuring more than one fluorescence quenching curve is highly advantageous. That is, even application of the established procedure for assessing the fluorescence quantum yield [7] to a single curve may lead to discrepancy in the determined parameters.…”

“…That is, even application of the established procedure for assessing the fluorescence quantum yield [7] to a single curve may lead to discrepancy in the determined parameters. On the other hand, the use of some advanced algorithms [14][15] requires reaching a saturation in quenching, i.e. the use of high ligand concentrations, that has certain shortcomingse.g.…”

“…The question about the possibility to J of Biomedical Photonics & Eng 6 222 Jun 2020 © J-BPE 020303-5 obtain the information about stoichiometry using fluorescence methods was reviewed in detail in Refs. [14,15], and as it was already mentioned, for determination of the exact number of binding sites it is crucial to measure several titration curves with ligand concentrations up to the values that significantly exceed 1/! !…”

“…! [14,15]. In this work we propose the method for simple FQT data processing in the region of parameters, where saturation is not achieved, to test the hypotheses whether a single binding site (!…”

“…A number of methods was suggested in the literature, which allow estimation of the number of binding sites from titration curves [13][14][15]. Nevertheless, these methods require measurements of several titration curves in the regime when concentration of ligand must be high and significantly exceeds the characteristic dissociation constant of a complex [14][15], that is sometimes hardly possible due to experimental artifacts like the inner filter effect or low quantities of the ligand available. Hence, a simple and reliable test to assess the binding stoichiometry in more realistic experimental conditions is still of interest.…”