Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence is the dye molecules self-quenching, which accompanies resonance radiationless excitation energy transfer.

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils.

The fluorescence of the benzothiazole dye thioflavin T (ThT) is a well-known test for amyloid fibril formation. It has now become evident that ThT can also be used for structural investigations of amyloid fibrils and even for the treatment of amyloid diseases. In this case, one of the most urgent problems is an accurate determination of ThT-amyloid fibril binding parameters: the number of binding modes, stoichiometry, and binding constant for each mode. To obtain information concerning the ThT-amyloid fibril binding parameters, we propose to use absorption spectrophotometry of solutions prepared by equilibrium microdialysis. This approach is inherently designed for the determination of dye-receptor binding parameters. However, it has been very rarely used in the study of dye-protein interactions and has never been used to study the binding parameters of ThT or its analogues to amyloid fibrils. We showed that, when done in corpore, this approach enables the determination of not only binding parameters but also the absorption spectrum and molar extinction coefficient of ThT bound to sites of different binding modes. The proposed approach was used for the examination of lysozyme amyloid fibrils. Two binding modes were found for the ThT-lysozyme amyloid fibril interaction. These binding modes have significantly different binding constants (K(b1) = 7.5 × 10(6) M(-1), K(b2) = 5.6 × 10(4) M(-1)) and a different number of dye binding sites on the amyloid fibrils per protein molecule (n(1) = 0.11, n(2) = 0.24). The absorption spectra of ThT bound to sites of different modes differ from each other (ε(b1,max) = 5.1 × 10(4) M(-1) cm(-1), ε(b2,max) = 6.7 × 10(4) M(-1)cm(-1), λ(max) = 449 nm) and significantly differ from that of free ThT in aqueous solution (ε(max) = 3.2 × 10(4) M(-1)cm(-1), λ(max) = 412 nm).

Amyloids are protein aggregates with a highly ordered spatial structure giving them unique physicochemical properties. Different amyloids not only participate in the development of numerous incurable diseases but control vital functions in archaea, bacteria and eukarya. Plants are a poorly studied systematic group in the field of amyloid biology. Amyloid properties have not yet been demonstrated for plant proteins under native conditions in vivo. Here we show that seeds of garden pea Pisum sativum L. contain amyloid-like aggregates of storage proteins, the most abundant one, 7S globulin Vicilin, forms bona fide amyloids in vivo and in vitro. Full-length Vicilin contains 2 evolutionary conserved β-barrel domains, Cupin-1.1 and Cupin-1.2, that self-assemble in vitro into amyloid fibrils with similar physicochemical properties. However, Cupin-1.2 fibrils unlike Cupin-1.1 can seed Vicilin fibrillation. In vivo, Vicilin forms amyloids in the cotyledon cells that bind amyloid-specific dyes and possess resistance to detergents and proteases. The Vicilin amyloid accumulation increases during seed maturation and wanes at germination. Amyloids of Vicilin resist digestion by gastrointestinal enzymes, persist in canned peas, and exhibit toxicity for yeast and mammalian cells. Our finding for the first time reveals involvement of amyloid formation in the accumulation of storage proteins in plant seeds.

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).