Fig 1.
Absorption spectra of thioflavin T (ThT) incorporated into Sup35p amyloid fibrils.
Curves 1 and 2 represent the absorption spectra of ThT in chamber #1 (free ThT at concentration Cf) and in chamber #2 (superposition of the absorption spectra of free ThT at concentration Cf, ThT bound to fibrils at concentration Cb, and the apparent absorption caused by the light scattering) after equilibrium was attained. Curve 3 represents the optical density caused by the fibril light scattering as calculated by the equation Ascat = aλ-m. The coefficients a and m were determined from the linear part of curve 2 (where there is no active dye absorption) plotted in logarithmic coordinates lg(Ascat) = f(lg(λ)) (see Insert, curve 3). Curve 4 represents the total absorption of free and bound dye after light scattering subtraction (A(λ)#2 –Dscat). Curve 5 is the absorption spectrum of ThT incorporated in the amyloid fibrils evaluated as Ab(λ) = A(λ)#2 –A(λ)scat−A(λ)#1 (the difference between spectra 4 and 1). Curve 6 is the absorption spectrum of the free dye at a concentration equal to that of bound dye (A(λ)0 – 2A(λ)#1).
Fig 2.
Scatchard plot for the ThT interaction with Sup35p amyloid fibrils.
The experimental data (circles) and best fit curve with the binding constants (Kbi) and the number of binding sites (ni) are shown.
Table 1.
Characteristics of thioflavin t bound to amyloid fibrils and free dye in aqueous solution.
Fig 3.
(a) Sup35p, (b) beta-2—microglobulin, (c) insulin, and (d) lysozyme amyloid fibrils.
Fig 4.
Determination of the molar extinction coefficient of ThT bound to Sup35p amyloid fibrils.
(a) Concentration of ThT bound to amyloid fibrils (Cb) as a superposition of the concentrations of dye bound to mode 1 (Cb1) and mode 2 (Cb2). (b) 3D dependence of Ab = Ab1 + Ab2 = εb1Cb1l + εb2Cb2l on Cb1 and Cb2. The experimental data and the values of the molar extinction coefficients εb1 and εb2 obtained by multiple nonlinear regression are presented. (c) Absorption spectra of ThT for mode 1 and mode 2 of the binding to Sup35p amyloid fibrils in units of the molar extinction coefficient.
Fig 5.
Determination of the fluorescence quantum yield of ThT bound to Sup35p amyloid fibrils.
(a) The dependencies of the experimentally recorded fluorescence intensity (F) and corrected fluorescence intensity (F0 = F/W) of ThT bound to fibrils on its absorption (Ab). Fluorescence measurements were conducted using a Cary Eclipse spectrofluorimeter (Varian, Australia). (b) 3D dependence of F0 = Ab1qb1 + Ab2qb2 on Ab1 and Ab2. The experimental data and the values of the fluorescence quantum yields qb1 and qb2 obtained by multiple nonlinear regression are presented.