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Binding Stoichiometry and Affinity of Fluorescent Dyes to Proteins in Different Structural States

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Intrinsically Disordered Protein Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 895))

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.

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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.

Author information

Authors and Affiliations

  1. Laboratory of Structural Dynamics, Stability and Folding of Proteins of the Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia

    Anna I. Sulatskaya, Olga I. Povarova, Irina M. Kuznetsova & Konstantin K. Turoverov

  2. Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL, USA

    Vladimir N. Uversky

  3. Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russia

    Vladimir N. Uversky

Authors
  1. Anna I. Sulatskaya
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  2. Olga I. Povarova
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  3. Irina M. Kuznetsova
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  4. Vladimir N. Uversky
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  5. Konstantin K. Turoverov
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Corresponding author

Correspondence to Vladimir N. Uversky .

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Editors and Affiliations

  1. Department of Molecular Medicine, College of Medicine, University of South Florida, Bruce B. Downs Blvd 12901, Tampa, 33612, Florida, USA

    Vladimir N. Uversky

  2. Dept. Biochemistry & Molecular Biology, Center for Computational Biology &, Indiana University, West 10th Street 410, Indianapolis, 46202, Indiana, USA

    A. Keith Dunker

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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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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-926-6

  • Online ISBN: 978-1-61779-927-3

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