ИСТИНА

Amyloid fibrils formation accompanies a range of serious disorders such as Alzheimer’s and Parkinson’s diseases, diabetes and prion diseases, etc. Fluorescent dye thioflavin T (ThT) is widely used for detection of amyloid fibrils presence, examination of their structure and kinetics of formation. However, the photophysical properties of ThT still remain under discussion. Along with the already commonly accepted notion that ThT binds to amyloid fibrils in monomeric form, there are publications in which it is suggested that fluorescence of ThT bound to amyloid fibrils is caused by dimers, excimers or even micelles of dye molecules. Our work was aimed to clarify the reasons of dramatic changes in fluorescent properties of ThT when it binds to amyloid fibrils and to analysis of the existing models of dye incorporation into fibrils. In this work we used absorption and fluorescent spectrometry. Fluorescence of ThT aqueous solutions with high absorbance was determined using Cary Eclipse spectrofluorimeter and a new method which we elaborated for correction of recorded fluorescence intensity on the inner filter effect. We showed that ThT can form excimers at high concentration in aqueous solution. However the comparison of fluorescence spectra of ThT in monomer and excimer forms in aqueous solution with that of ThT incorporated into amyloid fibrils allowed to conclude that fluorescence of ThT bound to fibrils is determined solely by its monomeric form. The increase in dye fluorescence intensity on its binding to amyloid fibrils can be explained by ThT molecular rotor nature. The results of this work are important for successful use of ThT for the investigation amyloid fibrils structure and, in particular, for determination of parameters of ThT-amyloid fibrils binding.