https://doi.org/10.1140/epjst/e2011-01513-1
Regular Article
Binding of Biotin to Streptavidin: A combined fluorescence correlation spectroscopy and time-resolved fluorescence study
1 Department of Applied Physics, Kungliga Tekniska Högskolan, 100 44 Stockholm, Sweden
2 Dipartimento di Fisica e Matematica, Università degli Studi dell’Insubria and C.N.I.S.M., via Valleggio 11, 22100 Como, Italy
3 Department of Physics and Astronomy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands
4 Department of Physics, University of Fribourg, Chemin du Musée 3, 1700 Fribourg, Switzerland
5 Centre for Quantum Photonics, H. H. Wills Physics Laboratory and Department of Electrical and Electronic Engineering, University of Bristol, Bristol, UK
6 INRS-EMT Université du Québec, 1650 Blvd. Lionel Boulet, Varennes, Quebec J3X 1S2, Canada
7 Istituto di Fotonica e Nanotecnologie, Consiglio Nazionale delle Ricerche and C.N.I.S.M., via Valleggio 11, 22100 Como, Italy
8 Department of Medical Biophysics, Karolinska Institutet, 17 177 Stockholm, Sweden
9 Laboratory of Biomedical Optics, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland
a e-mail: luca.nardo@uninsubria.it
Received:
29
August
2011
Revised:
4
October
2011
Published online:
7
December
2011
The Biotin-Streptavidin complex is a widely studied system in biology and biophysics, because of its extremely strong non-covalent binding affinity. The latter is often exploited to link molecules to substrates or to one another. However, the details of the Biotin-Streptavidin binding have not been fully elucidated so far. Particularly, the role of cooperative effects in enhancing the binding affinity has not been clarified. Our long-term aim is to investigate this point by implementing two complementary approaches, fluorescence correlation spectroscopy and time-correlated single-photon counting. As both methods rely on the analysis of fluorescence signals, biotin labeled with Atto-550-dye was used. In this work, in order to get a first overview of the system, we analyzed solutions in three paradigmatic ranges of Biotin-to-Streptavidin concentration ratio. Fluorescence correlation spectroscopy measurements allowed us to extract diffusion times of free biotin and of biotin-Streptavidin complexes, and also to gain information about the dynamics of the intersystem crossing between the first excited triplet and the first excited singlet states. Time-correlated single-photon counting made it possible to derive the lifetimes of the different species in solution, as well as to deduce relevant information about the relative abundance of Streptavidin-complexed and free Biotin.
© EDP Sciences, Springer-Verlag, 2011
