Figure 1.
Absorption spectra of thioflavin T (ThT) incorporated in the amyloid fibril.
Curves 1 and 2 represent absorption spectra of ThT in chamber #1 (free ThT at concentration Cf) and in chamber #2 (superposition of the absorption spectra of free ThT in concentration Cf, ThT bound to fibril in concentration Cb, and the apparent absorption caused by the light scattering) after the equilibrium attainment. Curve 3 represents optical density determined by the fibril light scattering as calculated by the equation Dscat = aλ−m. Coefficients a and m were determined from the linear part of the curve 2 (where there is no active dye absorption) plotted in logarithmic coordinates lg(Dscat) = f(lg(λ)) (see Insert, curve 3). Curve 4 represents the total absorption of free and bound dye after light scattering subtraction (D(λ)#2−Dscat). Curve 5 is the absorption spectra of ThT incorporated in the amyloid fibril evaluated as Db(λ) = D(λ)#2−D(λ)scat−D(λ)#1 (the difference between the spectra 4 and 1). Curve 6 is the absorption spectrum of the free dye at the concentration equal to that of bound dye (D(λ)0−2D(λ)#1). This curve allows for the evaluation of the change in the molar extinction coefficient of ThT when bound to fibril. Concentration of protein that was used for amyloid fibril preparation was Cp = 6.9⋅10−5 M.
Figure 2.
Scatchard plots for ThT interaction with insulin amyloid fibril.
Experimental data (circles) and best fit curve with binding constants (Kbi) and number of binding sites (ni) given on the panels.
Table 1.
Characteristics of thioflavin T bound to amyloid fibrils, acetylcholinesterase and free dye in water solution.
Figure 3.
Determination of the molar extinction coefficient of thioflavine T (ThT) bound to insulin amyloid fibril.
(a) Concentration of ThT, bound with amyloid fibril (Cb), as superposition of the dye concentrations bound to mode 1 (Cb1) and mode 2 (Cb2). (b) The dependence Db = Db1+Db2 = εb1Cb1 l+εb2Cb2 l. In the panel experimental data, best fit curve and the values of molar extinction coefficients εb1 and εb2 obtained by multiple nonlinear regression are presented. (c) Absorption spectra of ThT, bound to mode 1 and mode 2 in the units of the molar extinction coefficient.
Figure 4.
The dependence of fluorescence intensity on optical density of fluorophore and on total optical density of solution.
(a) The dependences of fluorescence intensity on optical density (DATTO) of the fluorescence dye ATTO-425 with known quantum yield (qATTO = 0.9) calculated from equation (15) and experimentally recorded I. The dependence of fluorescence intensity on the optical density of the fluorescent substance (I) differs from the calculated one (Icalc) because an increase in the total optical density of the solution results in the increased absorption of the excitation light by the solution layers adjacent to the front wall of the spectrofluorometer cell, while the detecting system of spectrofluorometer “see” the central part of the cell, which is reached by a respectively smaller part of the excitation light. Due to this effect, the recorded fluorescence intensity begins to diminish after optical density reaches some value. The discussed effects depend on the instrument used and must also be taken into account. This can be accomplished by replacing Wcalc with W, which depends on total optical density of the solution and can be determined experimentally. As for Wcalc, the value of W is determined only by the total optical density of solution and does not depend on the contribution of the optical density of the fluorescent substance. The dependences of Wcalc and W on total optical density are given in the Insert. The strait dashed line is the dependence of DATTOq on DATTO calculated as DATTOq = Icalc/Wcalc = I/W. (b) The dependencies of the experimentally recorded fluorescence intensity (I) and the reduced fluorescence intensity (I/W) of ThT bound to insulin fibril on its optical density (Db). The strait dashed line is the dependence of DATTOq on DATTO for the fluorescence dye ATTO-425 (etalon with qATTO = 0.9).
Figure 5.
Determination of the fluorescence quantum yield of thioflavine T (ThT) bound to fibril.
(a) Optical density of ThT bound to amyloid fibril as superposition of optical densities of the dye bound in mode 1 (Db1) and mode 2 (Db2). (b) 3D dependence of Db1qb1+Db2qb2 from Db1 and Db2. Experimental data, best fit curve and the values of qb1 and qb2, determined multiple nonlinear regression are presented. (c) Reduced fluorescence intensities of ThT/W as superposition of the reduced fluorescence intensities of the dye bound to mode 1 (Db1qb1) and mode 2 (Db2qb2).