Abstract
A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates.
Highlights
Benzothiazole dye thioflavin T (ThT) fluorescence is a wellknown test for amyloid fibril formation in diseases associated with disturbances in protein folding, such as Alzheimer’s and Parkinson’s diseases, type II diabetes, and prion diseases
The study of the spectral properties of ThT [5] and the dependence of its fluorescence quantum yield on solution temperature and viscosity [6,7,8], together with quantum-chemical calculations of the dye molecule in the ground and excited states [9], have led us to believe that the significant increase in the fluorescence quantum yield when the dye is incorporated into the amyloid fibril is caused by a restriction of the torsion oscillations of ThT fragments relative to each other in the excited state [7,8]
In almost all of the works focused on this problem, the binding constants were evaluated on the basis of the dependence of ThT fluorescence intensity on either ThT or fibril concentration
Summary
Benzothiazole dye thioflavin T (ThT) fluorescence is a wellknown test for amyloid fibril formation in diseases associated with disturbances in protein folding, such as Alzheimer’s and Parkinson’s diseases, type II diabetes, and prion diseases. This approach is based on the unique property of ThT to form highly fluorescent complexes with amyloid and amyloid-like fibril [1,2]. In almost all of the works focused on this problem, the binding constants were evaluated on the basis of the dependence of ThT fluorescence intensity on either ThT or fibril concentration The results of these studies have been summarized in a review by Groenning [18]
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