Quantitative analysis of the benzanthrone aminoderivative binding to amyloid fibrils of lysozyme
Abstract
The accumulation of amyloid fibrils in different tissues is associated with a number of neurodegenerative
diseases. Despite a huge variety of amyloid-specific probes, all of them suffer from many drawbacks,
highlighting the necessity of searching for more preferable dyes. In the present work, the potential of new
fluorescent probe AM3 for selective detection of fibrillar protein aggregates, formed from lysozyme, has
been evaluated. To quantify the affinity of this dye for amyloid fibrils, the isotherms of dye binding to the
fibrillar lysozyme have been derived from fluorimetric titration. Parameters of the dye-protein
complexation: association constant, molar fluorescence, and binding stoichiometry, calculated from the
Langmuir adsorption model revealed that AM3 interacts strongly with protein insoluble aggregates. High
values of the binding parameters make AM3 an alternative to a widely-used amyloid-specific probe
Thioflavin T. We also investigated the effects of polarity and viscosity on AM3 fluorescence properties.
The binding of AM3 to the protein hydrophobic cavities has been followed by the redshift of the dye
emission spectra, which can be explained by H-bonding between proton-donating groups of the protein
and carbonyl moiety of the probe. A long-wavelength shift of emission maximum was observed also upon
increasing the excitation wavelength. This finding suggests that the reorientation time of solvent molecules is higher than the dye fluorescence lifetime. Fluorescence anisotropy studies revealed slow rotation
diffusion of the probe, bound to amyloid fibrils being indicative of high viscosity of AM3
microenvironment. The observed photophysical properties of the new aminobenzanthrone derivative
make AM3 a perspective probe for basic research and medical diagnostics.
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References
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