Binding Stoichiometry and Affinity of Fluorescent Dyes to Proteins in Different Structural States
Fluorescent Dyes, ANS, Binding Stoichiometry, Binding Affinity, Bovine Serum Albumin, Equilibrium Microdialysis, Fluorescence Intensity, Fluorescence Quantum Yield
Digital Object Identifier (DOI)
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.
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Citation / Publisher Attribution
Binding Stoichiometry and Affinity of Fluorescent Dyes to Proteins in Different Structural States, in V. N. Uversky & A. K. Dunker (Eds.), Intrinsically Disordered Protein Analysis, Humana Press, p. 441-460
Scholar Commons Citation
Sulatskaya, Anna I.; Povarova, Olga I.; Kuznetsova, Irina M.; Uversky, Vladimir N.; and Turoverov, Konstantin K., "Binding Stoichiometry and Affinity of Fluorescent Dyes to Proteins in Different Structural States" (2012). Molecular Medicine Faculty Publications. 614.