Department of Nuclear and Medical Physics, V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv 61022, Ukraine. Department of Neuroscience and Biomedical Engineering, School of Science and Technology, Aalto University, FI-00076, Espoo, Finland. Author to whom any correspondence should be addressed. 19/2 Tankopiya Str., ap. 47, Kharkov 61091, Ukraine.
Methods Appl Fluoresc. 2016 Sep 6;4(3):034010. doi: 10.1088/2050-6120/4/3/034010.
Fluorescence represents one of the most powerful tools for the detection and structural characterization of the pathogenic protein aggregates, amyloid fibrils. The traditional approaches to the identification and quantification of amyloid fibrils are based on monitoring the fluorescence changes of the benzothiazole dye thioflavin T (ThT) and absorbance changes of the azo dye Congo red (CR). In routine screening it is usually sufficient to perform only the ThT and CR assays, but both of them, when used separately, could give false results. Moreover, fibrillization kinetics can be measured only by ThT fluorescence, while the characteristic absorption spectra and birefringence of CR represent more rigid criteria for the presence of amyloid fibrils. Therefore, it seemed reasonable to use both these dyes simultaneously, combining the advantages of each technique. To this end, we undertook a detailed analysis of the fluorescence spectral behavior of these unique amyloid tracers upon their binding to amyloid fibrils from lysozyme, insulin and an N-terminal fragment of apolipoprotein A-I with Iowa mutation. The fluorescence measurements revealed several criteria for distinguishing between fibrillar and monomeric protein states: (i) a common drastic increase in ThT fluorescence intensity; (ii) a sharp decrease in ThT fluorescence upon addition of CR; (iii) an appearance of the maximum at 535-540 nm in the CR excitation spectra; (iv) increase in CR fluorescence intensity at 610 nm. Based on these findings we designed a novel combined ThT-CR fluorescence assay for amyloid identification. Such an approach not only strengthens the reliability of the ThT assay, but also provides new opportunities for structural characterization of amyloid fibrils.
荧光代表了用于检测和结构表征致病蛋白聚集体（淀粉样纤维）的最有力的工具之一。传统的淀粉样纤维鉴定和定量方法基于监测苯并噻唑染料硫黄素 T（ThT）的荧光变化和偶氮染料刚果红（CR）的吸光度变化。在常规筛选中，通常只需要进行 ThT 和 CR 测定，但它们单独使用时都可能给出错误的结果。此外，只有 ThT 荧光才能测量纤维化动力学，而 CR 的特征吸收光谱和双折射则代表了淀粉样纤维存在的更严格的标准。因此，同时使用这两种染料似乎是合理的，结合每种技术的优势。为此，我们详细分析了这些独特的淀粉样示踪剂在与溶菌酶、胰岛素和载脂蛋白 A-I 的 N 端片段（爱荷华突变）的淀粉样纤维结合时的荧光光谱行为。荧光测量揭示了区分纤维状和单体蛋白状态的几个标准：（i）ThT 荧光强度的普遍急剧增加；（ii）CR 加入后 ThT 荧光的急剧下降；（iii）在 CR 激发光谱中出现 535-540nm 的最大值；（iv）CR 荧光强度在 610nm 处增加。基于这些发现，我们设计了一种新的 ThT-CR 联合荧光测定法用于淀粉样鉴定。这种方法不仅增强了 ThT 测定的可靠性，而且为淀粉样纤维的结构表征提供了新的机会。
