Abstract
Protocol of determination of binding stoichiometry and affinity of fluorescent dyes with proteins in different structural states is proposed. The proposed approach is based on the spectrophotometric determination of concentrations of dye bound to protein and free dye in solutions prepared by equilibrium microdialysis. This technique allows also determining spectral properties of the bound dyes. The restrictions of the use of dye fluorescence intensity for characterization of its interaction with the target protein are discussed. It is shown that the dependence of the dye fluorescence intensity on its optical density together with the data on its binding parameter can give information about the dye fluorescence quantum yield. All procedures are illustrated by interaction of 8-anilino-1-naphthalenesulfonate (ANS) with bovine serum albumin.
Key words
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Abbreviations
- ANS:
-
8-Anilino-1-naphthalenesulfonate
- BSA:
-
Bovine serum albumin
- ThT:
-
Thioflavin T
- QS:
-
Quinine sulfate
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Acknowledgement
The work was partially supported by Program Molecular and Cellular Biology of the Russian Academy of Science.
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Sulatskaya, A.I., Povarova, O.I., Kuznetsova, I.M., Uversky, V.N., Turoverov, K.K. (2012). Binding Stoichiometry and Affinity of Fluorescent Dyes to Proteins in Different Structural States. In: Uversky, V., Dunker, A. (eds) Intrinsically Disordered Protein Analysis. Methods in Molecular Biology, vol 895. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-927-3_26
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DOI: https://doi.org/10.1007/978-1-61779-927-3_26
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