Effect of acidic and basic pH on Thioflavin T absorbance and fluorescence

Abstract

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.

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

“…Below pH 3, protonation of the tertiary amine (Atom c in Figure 5(A)) leads to a decrease in ThT absorption and emission [94]. Caution should be exercised when comparing in situ ThT intensities at acidic and neutral pH values, since the lower emission of protonated ThT can be mistaken for lower fibril load.…”

Intrinsic Turn‐On Response of Thioflavin T in Complexes

“…Complexes with certain macrocycles, oligonucleotides, and other non‐amyloid‐forming proteins also show substantial turn‐on responses. Fluorescence of ThT is also sensitive to other environmental factors including temperature, pressure, solvent viscosity, and pH . A better understanding of how these various factors affect ThT photophysics will improve design and utility of solution assays involving ThT.…”

“…Fluorescence of ThT is also sensitivet oo ther environmentalf actors including temperature, pressure, solventv iscosity,a nd pH. [10][11][12][13][14][15] Ab etter understanding of how these various factors affect ThT photophysics will improve designa nd utility of solution assays involving ThT.F or example, it is not clear whether ThT can be used to distinguish between prefibrillar toxic oligomers of amyloidogenic peptidesa nd proteins or their fibril forms.T he ability to selectively sense oligomerso fa certain size or conformation may be important as these may represent an important therapeutic target. Here, we study the photophysics of ThT with and without individual binding part-ners inside am ass spectrometer, an environmentt hat is free of interactions with solventm olecules, andw here the presence of binding partners( e.g.,T hT dimers) is readily identifiable from the mass spectrum.…”

High resolution structure of a partially folded insulin aggregation intermediate

“…The aliquots were diluted in 10 mM phosphate buffer (pH 7.4) with 100 mM NaCl. 16,64 The ThT fluorescence was measured in a quartz cuvette; the spectrofluorometer (PTI) was set at an excitation wavelength of 450 nm, and the emission was observed at 482 nm.…”