ИСТИНА

One of the most widely and effectively used methods for the diagnostics of the formation and study of the structure of amyloid fibrils, accumulation of which accompanies many serious diseases (Alzheimer's disease, Parkinson's disease and others) is investigation of their interaction with fluorescent probes, and in particular, with thioflavin T (ThT). It is due to the fact that the interaction of this dye with amyloid fibrils is very specific. ThT does not interact with globular proteins in their native state, amorphous aggregates or oligomers. At the same time, the fluorescence quantum yield of the dye, which is equal to 0.0001 in aqueous solution, can increase by tens of thousands times, while ThT incorporates into amyloid fibrils. It should be noted that the current understanding of the mechanisms of ThT interaction with fibrils was formed on the basis of in vitro investigations in diluted aqueous solutions. These conditions significantly differ from the real conditions, in which proteins exist and operate in the cell (conditions of macromolecular crowding), where the concentration of biological macromolecules is in the range of 80-400 mg/ml and the occupied volume reaches 40% of the cell volume. Macromolecular crowding can have noticeable influence on the ability of proteins to form amyloid fibrils and the ways of their interactions with potential diagnostic agents, as well as on the properties of fluorescent probes. Thus, investigation of photophysical properties of ThT under macromolecular crowding conditions became the aim of the present work. Macromolecular crowding is typically mimicked by concentrated solutions of various inert polymers that serve as model “crowding agents”. Various crowding-agents: Dextran, PEG12000 and PEG400 (MW=70000, 12000, 400 Da respectively), with a wide range of concentrations were used in our study. In addition glycerol (MW=72 Da) was used as a low molecular weight agent for creating microviscosity conditions. It was shown that the absorption and the fluorescence intensity of the crowding agents do not contribute significantly to the absorption and fluorescence intensity of the dye. It was established that the presence of crowding agents and glycerol in the solution in concentration 400 mg/ml or 40% leads to a red shift of the absorption (from 5 to 9 nm) and fluorescence (from 3 to 7 nm) spectra of ThT, as well as to a slight increase in molar extinction coefficient (~ 1.1-1.2 times) and fluorescence quantum yield (~ 5-7 times). At the same time, it was shown that the change in photophysical properties of ThT induced by the presence of crowding agents with various molecular weight is significantly less than that induced by the presence of amyloid fibrils on the basis of insulin, lysozyme and beta-2-microglobulin. This confirms the possibility ThT using as a diagnostic agent in macromolecular crowding conditions.