Hackl Ellen V, Darkwah Joseph, Smith Geoff, Ermolina Irina
Leicester School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University, Leicester, UK.
Eur Biophys J. 2015 May;44(4):249-61. doi: 10.1007/s00249-015-1019-8. Epub 2015 Mar 22.
Thioflavin T (ThT) is a fluorescent dye able to enhance significantly its fluorescence quantum yield upon binding to protein amyloids. ThT assay is widely used to detect and quantify amyloids in a variety of conditions, including solutions with different pH levels. In the present work, the effect of acidic and basic pH on the conformation of the ThT molecule and its absorption and fluorescence properties was studied. The results show that both acidic and basic pH decrease significantly the intensity of ThT absorption in the visible region and fluorescence emission intensity. Low pHs induce an immediate "all-or-nothing" decrease in the ThT signal, while in alkaline solutions the ThT signal decreases gradually over time. pH-induced signal quenching is less in the presence of glycerol or protein aggregates. Two different mechanisms are responsible for the ThT signal quenching-the ThT hydroxylation at basic pH and protonation of the nitrogen atom of the dimethylamino group at acidic pH. ThT assays should be carefully carried out at basic or acidic pH as strong pH dependence of ThT could be responsible for misinterpretation and false positive/negative experimental results. The potential unsuitability of ThT as a probe in solutions with high pH (>9) has been shown.
硫黄素T（ThT）是一种荧光染料，与蛋白质淀粉样蛋白结合时能够显著提高其荧光量子产率。ThT检测法被广泛用于在各种条件下检测和定量淀粉样蛋白，包括不同pH值的溶液。在本研究中，研究了酸性和碱性pH对ThT分子构象及其吸收和荧光性质的影响。结果表明，酸性和碱性pH均显著降低了ThT在可见光区域的吸收强度和荧光发射强度。低pH值会导致ThT信号立即出现“全或无”的下降，而在碱性溶液中，ThT信号会随时间逐渐下降。在甘油或蛋白质聚集体存在的情况下，pH诱导的信号猝灭较少。ThT信号猝灭有两种不同的机制——碱性pH下的ThT羟基化和酸性pH下二甲基氨基氮原子的质子化。由于ThT对pH有很强的依赖性，可能会导致实验结果的误判和假阳性/阴性，因此在碱性或酸性pH下进行ThT检测时应格外小心。已证明ThT在高pH（>9）溶液中作为探针可能不合适。
