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

Area of Science:

• Biochemistry
• Neuroscience
• Molecular Biology

Background:

• Abnormal protein fibrillization is implicated in neurodegenerative diseases like Alzheimer's and Parkinson's, and type-II diabetes.
• The fluorescent dye Thioflavin T (ThT) is widely used to monitor protein fibril formation kinetics.
• Previous studies identified compounds causing false positives in ThT assays for Alzheimer's amyloid-β fibrils.

Purpose of the Study:

• To investigate whether specific compounds interfere with ThT fluorescence in insulin fibril formation.
• To determine if these interfering compounds act as true inhibitors of insulin fibrillization.

Main Methods:

• Utilized Thioflavin T (ThT) fluorescence assays to monitor insulin fibril formation kinetics.
• Tested the effect of selected compounds (Basic Blue 41, Basic Blue 12, Azure C, Tannic acid) on ThT fluorescence.
• Conducted kinetic experiments to assess the impact of compounds on fibrillization rate and lag-period.
• Verified the absence of soluble insulin post-experiment to rule out disaggregation.

Main Results:

• The tested compounds significantly decreased ThT fluorescence signal in the presence of insulin fibrils at submicromolar/micromolar concentrations.
• These compounds did not alter the fibrillization rate or increase the lag-period, unlike true inhibitors.
• The absence of soluble insulin confirmed that the compounds did not disaggregate pre-formed insulin fibrils.

Conclusions:

• Interference with the ThT assay by certain compounds is a general phenomenon, not limited to amyloid-β fibrils.
• The findings necessitate caution in interpreting kinetic data from ThT-based assays for protein fibrillization.
• Further validation is required to avoid misinterpreting compound effects on fibrillization kinetics.