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

Area of Science:

• Biochemistry
• Spectroscopy
• Chemical Biology

Background:

• Thioflavin T (ThT) is a fluorescent dye commonly used for amyloid detection.
• The ThT assay's reliability depends on consistent dye properties across various experimental conditions.
• Understanding pH effects on ThT is crucial for accurate amyloid quantification.

Purpose of the Study:

• To investigate the impact of acidic and basic pH on Thioflavin T (ThT) conformation, absorption, and fluorescence.
• To elucidate the mechanisms behind pH-induced changes in ThT signal intensity.
• To assess the suitability of ThT assays in solutions with extreme pH values.

Main Methods:

• Spectroscopic analysis of ThT conformation and fluorescence.
• Absorption and emission intensity measurements at varying pH levels.
• Comparative studies with and without glycerol or protein aggregates.

Main Results:

• Both acidic and basic pH significantly reduce ThT absorption and fluorescence intensity.
• Acidic pH causes an immediate decrease in the ThT signal, while alkaline pH leads to a gradual decline.
• ThT hydroxylation (basic pH) and protonation (acidic pH) are identified as quenching mechanisms.
• Glycerol or protein aggregates mitigate pH-induced signal quenching.

Conclusions:

• Extreme pH conditions significantly alter ThT properties, affecting assay reliability.
• ThT assays require careful execution at acidic or basic pH to avoid misinterpretation.
• ThT is potentially unsuitable as a probe in highly alkaline solutions (pH > 9).