The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds

Abstract

Thioflavin T (ThT) dye fluorescence is used regularly to quantify the formation and inhibition of amyloid fibrils in the presence of anti-amyloidogenic compounds such as polyphenols. However, in this study, it was shown, using three polyphenolics (curcumin, quercetin and resveratrol), that ThT fluorescence should be used with caution in the presence of such exogenous compounds. The strong absorptive and fluorescent properties of quercetin and curcumin were found to significantly bias the ThT fluorescence readings in both in situ real-time ThT assays and single time-point dilution ThT-type assays. The presence of curcumin at concentrations as low as 0.01 and 1 lm was sufficient to interfere with the ThT fluorescence associated with fibrillar amyloid-b(1-42) (0.5 lm) and fibrillar reduced and carboxymethylated j-casein (50 lm), respectively. The ThT fluorescence associated with fibrillar amyloid-b(1-42) was also biased using higher concentrations of resveratrol, a polyphenol that is not spectroscopically active at the wavelengths of ThT fluorescence, implying that there can be direct interactions between ThT and the exogenous compound and ⁄ or competitive binding with ThT for the fibrils. Thus, in all cases where ThT is used in the presence of an exogenous compound, biases for amyloid-associated ThT fluorescence should be tested, regardless of whether the additive is spectroscopically active. Simple methods to conduct these tests were described. The Congo red spectral shift assay is demonstrated as a more viable spectrophotometric alternative to ThT, but allied methods, such as transmission electron microscopy, should also be used to assess fibril formation independently of dye-based assays. Abbreviations CR, congo red; RCMj-CN, reduced and carboxymethylated j-casein; TEM, transmission electron microscopy; ThT, thioflavin T.

Myricetin Prevents Fibrillogenesis of Hen Egg White Lysozyme

“…However, since ThT fluorescence induction can be biased by the presence of exogenous compounds, 49 we also assessed the physical nature of myricetin fibril inhibition using TEM. Incubation of myricetin with HEWL resulted in the formation of amorphous aggregates, with little or no fibrils being observed ( Figure 4C,D).…”

Morin hydrate inhibits amyloid formation by islet amyloid polypeptide and disaggregates amyloid fibers

“…This may occur because they absorb strongly which leads to inner filter effects, or because they quench the bound thioflavin-T fluorescence, or because they inhibit thioflavin-T binding to amyloid. 48,49 The compounds under study here have the potential to interfere with thioflavin-T assays. Figure 2 shows the results of a thioflavin-T-monitored kinetic experiment in the presence of Myricetin.…”

“Clicked” Sugar–Curcumin Conjugate: Modulator of Amyloid-β and Tau Peptide Aggregation at Ultralow Concentrations

“…It should be noted that assays such as the thioflavin assay are not appropriate for testing anti-amyloidogenic properties of curcumin and its derivatives because the strong absorptive and fluorescent properties curcumin have been shown to significantly interfere with the thiofloflavin fluorescence readings. 29 To assess whether the enhanced bioefficacy of SC can be correlated with the antioxidant potential (AOP), we measured the AOP of curcumin, curcumin monoalkyne, and sugar− curcumin via the linoleic acid peroxidation method in accordance with a literature procedure. 30 SC showed higher AOP compared to both the curcumin and curcumin monoalkyne in water (see Supporting Information Figure S2).…”