Laboratory of Structural Dynamics, Stability and Folding of Proteins of the Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia.
J Phys Chem B. 2011 Oct 6;115(39):11519-24. doi: 10.1021/jp207118x. Epub 2011 Sep 14.
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).
苯并噻唑染料硫黄素 T（ThT）的荧光是一种常用于检测淀粉样纤维形成的方法。现在已经很明显，ThT 也可以用于淀粉样纤维的结构研究，甚至可以用于治疗淀粉样疾病。在这种情况下，最紧迫的问题之一是准确确定 ThT-淀粉样纤维结合参数：结合模式的数量、化学计量和每种模式的结合常数。为了获得有关 ThT-淀粉样纤维结合参数的信息，我们建议使用通过平衡微透析制备的溶液的吸收分光光度法。这种方法本质上是为了确定染料-受体结合参数而设计的。然而，它在研究染料-蛋白质相互作用中很少被使用，也从未被用于研究 ThT 或其类似物与淀粉样纤维的结合参数。我们表明，当在体内进行时，这种方法不仅可以确定结合参数，还可以确定与不同结合模式的结合部位结合的 ThT 的吸收光谱和摩尔消光系数。该方法用于检查溶菌酶淀粉样纤维。发现 ThT-溶菌酶淀粉样纤维相互作用存在两种结合模式。这些结合模式具有显著不同的结合常数（K(b1) = 7.5×10(6) M(-1)，K(b2) = 5.6×10(4) M(-1)）和每个蛋白质分子上与淀粉样纤维结合的染料结合位点数量不同（n(1) = 0.11，n(2) = 0.24）。与不同模式的结合部位结合的 ThT 的吸收光谱彼此不同（ε(b1,max) = 5.1×10(4) M(-1)cm(-1)，ε(b2,max) = 6.7×10(4) M(-1)cm(-1)，λ(max) = 449nm），并且与水溶液中的游离 ThT 显著不同（ε(max) = 3.2×10(4) M(-1)cm(-1)，λ(max) = 412nm）。
