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Interaction of novel benzanthrone derivative with amyloid lysozyme
(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Vus, K.O.; Trusova, V.M.; Gorbenko, G.P.; Zhytniakivska, O.A.; Kirilova, E.; Kirilov, G.; Kalnina, I.
A novel benzanthrone derivative AM18 was investigated with respect to its photophysical properties when bound to native, oligomeric and fibrillar hen egg white lysozyme. As shown by fluorimetric titration AM18 is more sensitive to pathogenic protein aggregates than Thioflavin T, however has no ability to differentiate between mature and immature lysozyme fibrils. The recovered affinity and fluorescence response of the novel probe to amyloid protein appeared to be similar to those of recently developed amyloid lysozyme-sensitive dyes like e. g. Nile Red and cyanine dye 7515. Despite the high increase of the probe emission in the presence of amyloid lysozyme compared to its fluorescence in buffer, the minimal amount that could be detected by 1 μM AM18 was 10 times lower for amyloid-native protein solutions due to high affinity of the dye for lysozyme monomers. In general, because of high quantum yields and “signal-to-noise” ratios in the presence of pathogenic protein aggregates AM18 appeared to be an effective tool for amyloid detection and characterization in vitro, being however unable to detect pathogenic protein aggregates in vivo like e.g. recently reported p-FTAA because of the sensitivity to lipids. Compared to previously reported AM3 a novel dye showed 2-fold lower “signal-to- noise” ratio in the presence of fibrillar lysozyme, and 2 fold lower blue shift of emission maximum. This tendency was explained in terms of decreased charge transfer from the donor to acceptor groupes of AM18 compared to AM3. Finally, as concluded from the comparison of AM18 and previously studied benzanthrone derivatives, the 5 nm – red edge excitation shift of AM18 is indicative of its possible binding to fibril “deep cavities”, containing no water. High anisotropy values of amyloid-bound dye led us to conclusion that the enhanced fluorescence of the probe is associated with the decrease of the rotational motion of the amino-substitute about the benzanthrone unit. This is a sign of AM18 behaviour as a molecular rotor.
Quantitative analysis of the benzanthrone aminoderivative binding to amyloid fibrils of lysozyme
(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Vus, K.O.; Trusova, V.M.; Gorbenko, G.P.; Kirilova, E.; Kirilov, G.; Kalnina, I.
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 red shift 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. Long-wavelength shift of emission maximum was observed also upon increasing the excitation wavelength. This finding suggests that 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.