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Application of Peak Intensity Analysis to Measurements of Protein Binding to Lipid Vesicles and Erythrocytes Using Fluorescence Correlation Spectroscopy: Dependence on Particle Size

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Abstract

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool for investigation of processes accompanied by changes in the mobility of molecules and complexes. In the present work, peak intensity analysis (PIA) in combination with the solution stirring using FCS setup was applied to explore the interaction between fluorescently labeled protein ligands and corresponding receptors located on membranes. In the system composed of biotinylated liposomes and fluorescently labeled streptavidin as a ligand, PIA allowed us to determine the optimum receptor concentration and demonstrate the essential dependence of the binding efficacy on the length of the linker between the biotin group and the polar head group of the lipid. The binding was dependent on the size of liposomes which was varied by lipid extrusion through filters of different pore diameters. The sensitivity of the method was higher with the liposomes of larger sizes. The PIA approach can be applied not only to liposomes but also to relatively large objects, e.g., erythrocytes or Sepharose beads derivatized with lactose as a receptor for the binding of viscumin and ricin.

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Abbreviations

FCS:

Fluorescence correlation spectroscopy

G(τ):

Autocorrelation functions

τ d :

Diffusion time

Stv:

Streptavidin

Rh-Stv:

Rhodamine-labeled streptavidin

Alexa-Stv, Rh-Stv:

Fluorescently-labeled streptavidin

Alexa-ricin:

Fluorescently-labeled Ricin

Alexa-viscumin:

Fluorescently-labeled viscumin

Lac-sepharose:

Sepharose beads derivatized with lactose

GM1:

Galactose-containing ganglioside

PE:

Phosphatidylethanolamine

Biotin-PE:

Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl)

Biotin-Cap-PE:

Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap biotinyl)

EggPC:

Egg yolk phosphatidylcholine

PBS:

Phosphate-buffered saline

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Acknowledgements

We are grateful to Alexander Vasiliev for the help in conducting several experiments, Wladas Kozlovsky, Liubov Safonova, and Victor Orlov for technical assistance. This work was financially supported by the Russian Foundation for Basic Research 16-34-01308 (to A.A.R., the isolation and purification of the lectins section), and the Russian Science Foundation 16-14-10025 (to Y.N.A., functional FCS measurements).

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Authors and Affiliations

  1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia, 119991

    Yuri N. Antonenko, Anna S. Lapashina & Elena A. Kotova

  2. Biological Department, Lomonosov Moscow State University, Moscow, Russia

    Alla A. Ramonova, Mikhail M. Moisenovich & Igor I. Agapov

  3. Shumakov Research Center of Transplantology and Artificial Organs, Moscow, Russia

    Igor I. Agapov

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  1. Yuri N. Antonenko
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  2. Anna S. Lapashina
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  3. Elena A. Kotova
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  5. Mikhail M. Moisenovich
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  6. Igor I. Agapov
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Correspondence to Yuri N. Antonenko.

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Antonenko, Y.N., Lapashina, A.S., Kotova, E.A. et al. Application of Peak Intensity Analysis to Measurements of Protein Binding to Lipid Vesicles and Erythrocytes Using Fluorescence Correlation Spectroscopy: Dependence on Particle Size. J Membrane Biol 250, 77–87 (2017). https://doi.org/10.1007/s00232-016-9938-6

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