Evaluation of ThT Augmentation and RLS Inner Filter Effect Caused by Highly Fluorescent Coumarin Derivative and Establishing It as True Inhibitor of Amyloid Fibrillation
Tht Biasing, Nile Red, Far-uv CD Kinetics, Inner Filter Effect, Fluorophore, Chromophore
Digital Object Identifier (DOI)
Screening of inhibitors that slow down or suppress amyloid fibrils formation relies on some simple but sensitive spectroscopy techniques. Thioflavin T (ThT) fluorescence assay is one of the most common, amyloid specific and sensitive method. However, if an inhibitor is itself fluorescent in the ThT fluorescence range, its screening becomes complicated and require complementary assays. One of such molecules, 6, 7-dihydroxycoumarin (6, 7-DHC, also known as aesculetin, esculetin, and cichorigenin) is fluorescent in the ThT emission range and absorbs in the ThT excitation range. Therefore, it can produce a subtractive effect attributed to primary inner filter effect and/or additive effect due to its self-fluorescence in ThT assay. Our study shows that 6, 7-DHC produces an additive effect in ThT fluorescence, which is minimized at high concentration of ThT and decrease in ThT fluorescence is solely due to its inhibitory effect against HSA fibrillation. These ThT fluorescence-based results are verified through other complementary assays, such as Rayleigh and dynamic light scattering and amyloid-specific Congo red binding assay. Furthermore, hydrophobicity reduction is studied through Nile red (NR) and kinetics through far-UV circular dichroism (far-UV CD) in place of the most commonly employed ThT assay owing to extremely high fluorescence of 6, 7-DHC during initial incubation period.
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Citation / Publisher Attribution
Archives of Biochemistry and Biophysics, v. 709, issue 30, art. 108981
Scholar Commons Citation
Masroor, Aiman; Chandel, Tajalli Ilm; Malik, Sadia; Mateen, Qazi Noorul; Uversky, Vladimir N.; and Khan, Rizwan Hasan, "Evaluation of ThT Augmentation and RLS Inner Filter Effect Caused by Highly Fluorescent Coumarin Derivative and Establishing It as True Inhibitor of Amyloid Fibrillation" (2021). Molecular Medicine Faculty Publications. 919.