Benzothiazole dye thioflavin T (ThT) is a sensitive probe for amyloid fibril detection. The ThT probing is based on its unique ability to form highly fluorescent complexes with amyloid and amyloid-like fibrils. In this work we propose an approach of ThT fluorescence quantum yield determination based on two key points: (1) fluorescence intensity (I) presentation as a multiple of two factors one of which the correcting factor (W) depends only on total optical density of solution, while the other is a multiple of optical density and fluorescence quantum yield of ThT bound to amyloid fibrils or their sum in the case of several binding modes (I = W Σ D(bi)q(i)) and (2) sample and reference solutions preparation by equilibrium microdialysis. The last allows to determine the values of optical densities of free (D(f)) and bound (D(b) = ΣD(bi)) dye. Thereafter, fluorescence quantum yield (q(bi)) of ThT bound to sites of i binding mode can be determined by multiple linear regression. The fluorescence quantum yield of ThT molecules bound to the sites of two binding modes of lysozyme amyloid fibrils with high and low binding constants (7.5 × 10(6) and 5.6 × 10(4) M(-1)) was found equal to 0.44 and 5 × 10(-4), respectively. The higher value of fluorescence quantum yield is larger than that for ThT in rigid isotropic solution (0.28), whereas the lower value is comparable to that of ThT in aqueous solution (1 × 10(-4)). At the same time absorption spectra of ThT bound to these modes coincide (450 nm) and are red-shifted in comparison with that of free ThT in aqueous solution (412 nm).
Research. Development. Production.
We are a leading supplier to the global Life Science industry with solutions and services for research, biotechnology development and production, and pharmaceutical drug therapy development and production.