The aim of the present work was investigation of the fluorescent dye thioflavin T (ThT) binding to acetylcholinesterase (AChE). ThT is an effective test for protease activity, as well as a probe for amyloid fibril formation. Despite the extended and active investigation of ThT–AChE binding, there is still no common view on the stoichiometry of this interaction. In particular, there is a hypothesis explaining the spectral properties of bound to AChE dye and high quantum yield of its fluorescence by formation of dimers or excimers of ThT. In order to confirm or deny this hypothesis, we proposed a new experimental approach for examination of ThT–AChE interaction based on spectroscopic investigation of samples prepared by equilibrium microdialysis. This approach allowed us to prove 1/1 ThT/AChE binding stoichiometry. The increase of ThT fluorescence quantum yield and lifetime accompanying its binding to AChE can be explained by the molecular rotor nature of this dye. Together with the coincidence of the positions of free and AChE-bound ThT fluorescence spectra, the obtained results prove the groundlessness of the hypotheses about ThT aggregation while binding to AChE. The model of ThT localization in the active site of AChE was proposed by using molecular docking simulations. These results also allowed us to suggest the key role of aromatic residues in ThT–AChE interaction, as observed for some amyloid fibrils.

中文翻译：

---

硫黄素T与乙酰胆碱酯酶的相互作用：平衡微透析制备的样品获得1：1结合化学计量的新证据。

本工作的目的是研究荧光染料硫黄素T（ThT）与乙酰胆碱酯酶（AChE）的结合。ThT是检测蛋白酶活性的有效方法，也是淀粉样蛋白原纤维形成的探针。尽管对ThT-AChE结合进行了广泛而积极的研究，但对于这种相互作用的化学计量仍然没有共识。特别是，有一个假设解释了与AChE染料结合的光谱特性，以及通过形成ThT的二聚体或准分子来获得其荧光的高量子产率。为了证实或否认这一假设，我们提出了一种新的实验方法，用于通过平衡微量渗析制备的样品的光谱研究来检查ThT-AChE相互作用。这种方法使我们能够证明1/1 ThT / AChE结合化学计量。ThT荧光量子产率的提高和伴随其与AChE结合的寿命的增加可以用这种染料的分子转子性质来解释。结合自由的和与AChE结合的ThT荧光光谱的位置相吻合，所获得的结果证明了关于ThT聚集与AChE结合的假说是毫无根据的。通过分子对接模拟，提出了ThT在AChE活性位点的定位模型。这些结果也使我们能够提出芳香族残基在ThT-AChE相互作用中的关键作用，如某些淀粉样蛋白原纤维所观察到的。获得的结果证明了关于ThT聚集与AChE结合的假说是毫无根据的。通过分子对接模拟，提出了ThT在AChE活性位点的定位模型。这些结果也使我们能够提出芳香族残基在ThT-AChE相互作用中的关键作用，如某些淀粉样蛋白原纤维所观察到的。获得的结果证明了关于ThT聚集与AChE结合的假说是毫无根据的。通过分子对接模拟，提出了ThT在AChE活性位点的定位模型。这些结果也使我们能够提出芳香族残基在ThT-AChE相互作用中的关键作用，如某些淀粉样蛋白原纤维所观察到的。