Interference of low‐molecular substances with the thioflavin‐T fluorescence assay of amyloid fibrils

Abstract

Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type-II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low-molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid-β fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag-period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes.

Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time–Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening

“…However, it is still unclear whether acridine inhibited PrP res and quenched ThT at the same time. It has been reported that, in dye-dependent seeded aggregation assays, low molecular weight substances can interfere with the fluorescence of dye compounds bound to amyloid fibrils [27]. Indeed, some compounds have been posited to compete with ThT for the same binding site on fibrils rather than directly inhibit the fibrilization process.…”

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

“…For example, the authors of (Noormägi et al 2012) showed that some low molecular weight substances (Tannic acid, Basic Blue 41, BB 12, Azure C) can interfere with the ThT assay by decreasing the fluorescence signal of ThT bound to insulin fibrils without inhibiting the insulin fibrillization process. The anionic, but not cationic or neutral, micellar microenvironment of SDS shifts the maximum of ThT absorption from 412 nm in buffer to 428 nm inside the micelle, with an increase in the peak molar absorptivity and a significant (some 13-fold) increase in ThT fluorescence (Kumar et al 2008).…”

Differential Modulating Effect of MoS2 on Amyloid Peptide Assemblies

“…These dyes interactw ith amyloid peptide in the aggregation process, which interferes with in situ measurements, for example Congo Red and Thioflavin T( ThT). [13] Herein, we combined label-free quartz crystal microbalance (QCM), atomic force microscopy (AFM), and circulard ichroism (CD) spectra,t oq uantitativelye xplore the modulating effect of MoS 2 on the fibrillization of two amyloid peptides, Ab (33-42), ak ey hydrophobic fragment of amyloid protein Ab (1-42), [14] and amylin (20)(21)(22)(23)(24)(25)(26)(27)(28)(29), the fibrillating core fragment of the humani slet amyloid polypeptide(hIAPP) involved in type-II diabetes. [15] We obtained MoS 2 suspensionsb yh ydrothermalt reatment of molybdic oxide and KSCN.…”