Absorption, fl uorescence, and fl uorescence excitation spectra of thiofl avin T in aqueous solutions and polyvinyl alcohol fi lms were studied. The dye aggregated when its concentration was increased. This was accompanied by a hypsochromic shift of the absorption spectrum and a bathochromic shift of the fl uorescence spectrum. It was shown that fl uorescence of the probe embedded in amyloid fi brils was caused by monomers rather than dimers or other aggregates.Introduction. Thiofl avin T (ThT) has recently been the subject of numerous investigations. This is due to the fact that it is used as a fl uorescent probe to study and detect amyloid fi brils [1-4], the formation of which parallels the development of several neurodegenerative diseases (Alzheimer′s, Parkinson′s, etc.). It is thought that ThT binds specifi cally to amyloid fi brils, after which its emission intensity increases by greater than three orders of magnitude [5]. Non-aggregated proteins in the solution typically have practically no effect on the probe fl uorescence [6].The fl uorescence properties of ThT, primarily the quantum yield, are known to be determined by the solvent rigidity or viscosity, which infl uence the ability and rate of torsional relaxation of the molecular fragments [7-9]. We proposed based on experimental data and quantum-chemical calculations that ThT is a so-called molecular rotor [10,11], for which the fl uorescence characteristics were observed to depend strongly on the solvent viscosity. Therefore, it was concluded that the reason for the signifi cant amplifi cation of the fl uorescence of aqueous solutions of ThT in the presence of fi brils was incorporation into a rigid micro-environment [5,12,13].Mechanisms of the incorporation of the probe into fi brils, the stoichiometry of the ThT-fi bril complex, and the issue of the specifi city of its interaction are still being discussed. The hypothesis of Krebs et al. seems to be the most convincing [14]. They noticed that side chains of amino acids within β-sheets of amyloid fi brils formed elongated strands along the fi bril axis, between which were elongated channels. According to their model [14], probe molecules were embedded in these channels so that its long axis was situated along the fi brils. ThT embedded in the channel was in close contact with amino-acid side chains of the β-sheets. This limited the rotational motion of the molecular fragments relative to each other. This caused the fl uorescence quantum yield to increase signifi cantly after its incorporation into the amyloid fi brils.It was hypothesized in several studies that ThT was incorporated into voids in the amyloid fi brils as dimers or excimers [15][16][17] or even micelles [18,19] so that the strong ThT fl uorescence with a maximum at 480-490 nm was emission from probe aggregates. According to the researchers, ThT aggregation caused a signifi cant bathochromic shift of the absorption spectrum (from 412 to ~445 nm) and a hypsochromic shift of the fl uorescence spectrum (from ~493 to 480 nm).The present wor...

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The absorption and fl uorescence spectra of a new styryl derivative of thiofl avin T 2-{(1E,3E)-4- in solvents with diff erent polarity and viscosity and also incorporated in the structure of amyloid fi brils and bovine serum albumin were investigated. A characteristic feature of the dye is an extremely low quantum yield of fl uorescence in low-viscosity solvents (10 -4 in water) which, however, increases signifi cantly in viscous solutions and when it is incorporated in the structure of proteins or amyloid fi brils. In the latter case the quantum yield increases by 8•10 3 times. On the basis of the experimental studies and quantum chemical calculations it was shown that Th-C23 exhibits the properties of a molecular rotor. The increase of the fl uorescence quantum yield in viscous solutions and in the biopolymers results from limitation of the torsional rotation of the molecular fragments, leading to fl uorescence quenching. The longwavelength location of the absorption spectrum and the fl uorescence spectrum of the new dye in the red region of the spectrum (520 and 600 nm) makes it possible to use it as a fl uorescent marker that is sensitive to the viscosity (hardness) of the microenvironment not only in vitro but also in vivo.