Evaluating the Number of Ligand Binding Sites on Protein from Tryptophan Fluorescence Quenching under Typical Experimental Conditions
Paper #3365 received 12 May 2020; accepted for publication 28 May 2020; published online 22 Jun 2020.
Fluorescence quenching technique is extensively applied for the characterization of intermolecular interactions in the solution that is one of the major problems in biochemistry and pharmacology. Using the Stern-Volmer equation, one can obtain a measure of binding affinity calculated under the assumption of static quenching, while the possibility to determine other binding parameters is under discussion. Several mathematical approaches are known, which allow determining the number of binding sites from fluorescence quenching curves. However, they usually require high concentrations of the ligand to obtain saturating binding curves that could be complicated in a number of experimental conditions. In this paper, we present a simple algorithm, which allows to prove that the number of binding sites in the system is equal to one or not and to verify that the quantum yield of the complex is zero. The advantage of the suggested approach is its applicability at typical conditions used in tryptophan fluorescence quenching experiments for the protein-ligand binding. A web interface for automated processing of fluorescence quenching experiments based on the suggested approach is presented.
1. M. E. Burton, L. M. Shaw, J. J. Schentag, and W. E. Evans (eds.), Applied pharmacokinetics & pharmacodynamics: principles of therapeutic drug monitoring, Lippincott Williams & Wilkins, Philadelphia, USA (2006).
2. D. L. Nelson, A. Lehninger, and M. M. Cox, Lehninger – principles of biochemistry, D. Nelson, M. B. Cox (Eds.), Macmillan, London, UK (2008).
3. K. Hirose, “A practical guide for the determination of binding constants,” Journal of inclusion phenomena and macrocyclic chemistry 39(3), 193–209 (2001).
4. J. R. Lakowicz (Ed.), Principles of fluorescence spectroscopy, Springer Science & Business Media, Berlin/Heidelberg, Germany (2013).
5. P. R. Callis, “Binding phenomena and fluorescence quenching. I: descriptive quantum principles of fluorescence quenching using a supermolecule approach,” Journal of Molecular Structure 1077, 14–21 (2014).
6. M. Van De Weert, “Fluorescence quenching to study protein-ligand binding: common errors,” Journal of fluorescence 20(2), 625–629 (2010).
7. M. Van de Weert, L. Stella, “Fluorescence quenching and ligand binding: A critical discussion of a popular methodology,” Journal of Molecular Structure 998(1–3), 144–150 (2011).
8. E. Lissi, C. Calderón, and A. Campos, “Evaluation of the number of binding sites in proteins from their intrinsic fluorescence: limitations and pitfalls,” Photochemistry and photobiology 89(6), 1413–1416 (2013).
9. T. N. Tikhonova, E. A. Shirshin, G. S. Budylin, V. V. Fadeev, and G. P. Petrova, “Assessment of the europium(III) binding sites on albumin using fluorescence spectroscopy,” The Journal of Physical Chemistry B 118(240), 6626–6633 (2014).
10. M. van de Weert, L Stella, “The dangers of citing papers you did not read or understand,” Journal of Molecular Structure 1186, 102–103 (2019).
11. Y. Hong, J. W. Lam, and B. Z. Tang, “Aggregation-induced emission,” Chemical Society Reviews 40(11), 5361–5388 (2011).
12. M. R. Eftink, Fluorescence methods for studying equilibrium macromolecule-ligand interactions, In Methods in enzymology 278, 221–225 (1997).
13. L. D. Ward, Measurement of ligand binding to proteins by fluorescence spectroscopy, Chapter in Methods in enzymology 117, 400–414 (1985).
14. W. Bujalowski, M. J. Jezewska, and P. J. Bujalowski, “Signal and binding. I. Physico-chemical response to macromolecule-ligand interactions,” Biophysical Chemistry 222, 7–24 (2017).
15. W. Bujalowski, M. J. Jezewska, and P. J. Bujalowski, “Signal and binding. II. Converting physico-chemical responses to macromoleculeligand interactions into thermodynamic binding isotherms,” Biophysical Chemistry 222, 25–40 (2017).
16. V. Kairys, L. Baranauskiene, M. Kazlauskiene, D. Matulis, and E. Kazlauskas, “Binding affinity in drug design: experimental and computational techniques,” Expert opinion on drug discovery 14(8), 755–768 (2019).
17. R. J. Leatherbarrow, “Using linear and non-linear regression to fit biochemical data,” Trends in biochemical sciences 15(12), 455–458 (1990).
18. Y.-Z. Zhang, X. Xiang, P. Mei, J. Dai, L.-L. Zhang, and Y. Liu, “Spectroscopic studies on the interaction of Congo Red with bovine serum albumin,” Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 72(4), 907–914 (2009).
19. H. M. C. de Paula, Y. L. Coelho, A. J. P. Agudelo, J. de Paula Rezende, G. M. D. Ferreira, G. M. D. Ferreira, A. C. dos Santos Pires, and L. H. M. da Silva, “Kinetics and thermodynamics of bovine serum albumin interactions with Congo red dye,” Colloids and Surfaces B: Biointerfaces 159, 737–742 (2017).
20. J. A. Hamilton, D. P. Cistola, J. D. Morrisett, J. T. Sparrow, and D. M. Small, “Interactions of myristic acid with bovine serum albumin: a 13C NMR study,” Proceedings of the National Academy of Sciences 81(12), 3718–3722 (1984).
21. E. L. Gelamo, C. H. T. P. Silva, H. Imasato, and M. Tabak, “Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling,” Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1594(1), 84–99 (2002).
22. I. M. Kuznetsova, A. I. Sulatskaya, O. I. Povarova, and K. K. Turoverov, “Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand–receptor binding characterization,” PLoS One 7(7), e40845 (2012).
© 2014-2020 Samara National Research University. All Rights Reserved.
Public Media Certificate (RUS). 12+