Thioflavins arefluorescent dyes that are available as at least two compounds, namelyThioflavin T andThioflavin S. Both are used forhistology staining andbiophysical studies of protein aggregation.[1] In particular, these dyes have been used since 1989 to investigate amyloid formation.[2] They are also used in biophysical studies of the electrophysiology of bacteria.[3] Thioflavins arecorrosive,irritant, and acutely toxic, causing serious eye damage.[4] Thioflavin T has been used in research intoAlzheimer's disease and otherneurodegenerative diseases.
When it binds tobeta sheet-rich structures, such as those in amyloid aggregates, the dye displays enhancedfluorescence and a characteristicred shift of itsemission spectrum.[5][6] Additional studies also considerfluorescence changes as result of the interaction with double stranded DNA.[7] This change in fluorescent behavior can be caused by many factors that affect theexcited statecharge distribution of thioflavin T, including binding to a rigid, highly-ordered nanopocket, and specific chemical interactions between thioflavin T and the nanopocket.[8][9]
Prior to binding to an amyloid fibril, thioflavin T emits weakly around 427 nm. Quenching effects of the nearby excitation peak at 450 nm is suspected to play a role in minimizing emissions.
When excited at 450 nm, thioflavin T produces a strong fluorescence signal at approximately 482 nm upon binding to amyloids. Thioflavin T molecule consists of a benzylamine and a benzothiazole ring connected through a carbon-carbon bond. These two rings can rotate freely when the molecule is in solution. The free rotation of these rings results in quenching of any excited state generated by photon excitation. However, when thioflavin T binds to amyloid fibrils, the two rotational planes of the two rings become immobilized and therefore, this molecule can maintain its excited state.[1]
Thioflavin T fluorescence is often used as a diagnostic of amyloid structure, but it is not perfectly specific for amyloid. Depending on the particular protein and experimental conditions, thioflavin T may[8] or may not[10] undergo a spectroscopic change upon binding to precursor monomers, small oligomers, unaggregated material with a highbeta sheet content, or evenalpha helix-rich proteins. Conversely, some amyloid fibers do not affect thioflavin T fluorescence,[11] raising the prospect offalse negative results.
Structure of thioflavin T bound to anamyloid-like oligomer ofβ2 microglobulin (in gray), in a complex that displays enhanced andred shifted fluorescence. Many factors that shift theexcited state charge from the dimethylaminobenzyl portion of thioflavin T (in blue) to the benzothiazole portion (in red), including binding to rigid, highly-ordered amyloid aggregates, can produce this 'positive' thioflavin T signal.[8]
Thioflavin S stain (left in green) andamyloid-Beta antibody immunocytochemistry (right) on adjacent sections of the hippocampus of a patient suffering fromAlzheimer's disease. Thioflavin S binds bothsenile plaques (SP) andneurofibrillary tangles (NFT), the two characteristic cortical lesions of Alzheimer's. Amyloid beta is a peptide derived from theamyloid precursor protein which is only found in senile plaques, and so only plaques are visible in the right hand image. The left image also has a red signal which exactly superimposes the green signal inlipofuscin granules (LP), which are autofluorescent inclusions derived fromlysosomes which accumulate in the human brain during normal aging.
In adultC. elegans, exposure to thioflavin T results "in a profoundly extended lifespan and slowed aging" at some levels, but decreased lifespan at higher levels.[12]
Thioflavin S is a homogenous mixture of compounds that results from the methylation of dehydrothiotoluidine withsulfonic acid. It is also used to stain amyloid plaques. Like thioflavin T it binds toamyloid fibrils but not monomers and gives a distinct increase in fluorescence emission. However unlike thioflavin T, it does not produce a characteristic shift in the excitation or emission spectra.[5] This latter characteristic of thioflavin S results in high background fluorescence, making it unable to be used in quantitative measurements of fibril solutions.[5] Another dye that is used to identify amyloid structure isCongo red.
^Biancardi A, Biver T, Mennucci B (2017). "Fluorescent dyes in the context of DNA-binding: The case of Thioflavin T".Int. J. Quantum Chem.117 (8): e25349.doi:10.1002/qua.25349.
