Thioflavin T hydroxylation at basic pH and its effect on amyloid fibril detection.
Vito Foderà1, Minna Groenning, Valeria Vetri
1Dipartimento di Scienze Fisiche e Astronomiche, Università degli Studi di Palermo, Palermo, Italy. fodera@fisica.unipa.it
The Journal of Physical Chemistry. B
|October 30, 2008
Summary
Thioflavin T (ThT) undergoes hydroxylation in alkaline solutions, affecting its use for amyloid detection. This study characterizes the process and its impact on quantitative fibril evaluation at basic pH.
Area of Science:
- Biophysical Chemistry
- Spectroscopy
- Chemical Biology
Background:
- Thioflavin T (ThT) is a widely used fluorescent dye for detecting amyloid fibrils in situ.
- Understanding ThT's behavior under varying conditions is crucial for accurate amyloid detection.
- The chemical stability and spectroscopic properties of ThT can be influenced by environmental factors like pH.
Purpose of the Study:
- To investigate the spectroscopic properties and chemical stability of Thioflavin T (ThT) in aqueous solutions.
- To characterize the hydroxylation process of ThT in alkaline conditions.
- To evaluate the impact of ThT hydroxylation on its efficiency for quantitative amyloid fibril detection at basic pH.
Main Methods:
- UV-vis absorption spectroscopy
- Proton Nuclear Magnetic Resonance (NMR) spectroscopy
- Density Functional Theory (DFT) calculations
- Fluorescence spectroscopy
Main Results:
- A reversible hydroxylation process of ThT was identified and characterized in alkaline solutions.
- The chemical structure of the hydroxylated ThT form was proposed based on spectroscopic and computational data.
- ThT hydroxylation was shown to affect the dye's performance in fluorescence spectroscopy for amyloid detection.
Conclusions:
- The hydroxylation of ThT in alkaline solutions is a significant factor influencing its spectroscopic properties.
- The proposed chemical structure provides a basis for understanding ThT's reactivity.
- These findings offer new insights into the efficiency and limitations of the ThT assay for quantitative amyloid fibril evaluation at basic pH.
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