Mechanism of thioflavin T binding to amyloid fibrils.
Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.
Khurana
Ritu
R
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA. rkhurana@ccmb.res.in
Coleman
Chris
C
Ionescu-Zanetti
Cristian
C
Carter
Sue A
SA
Krishna
Vinay
V
Grover
Rajesh K
RK
Roy
Raja
R
Singh
Shashi
S
D000682:Amyloid / Q000378:metabolism*; D052160:Benzothiazoles; D001665:Binding Sites; D003903:Deuterium; D005453:Fluorescence; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D018625:Microscopy, Atomic Force; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D008823:Micelles; D012996:Solutions; D013844:Thiazoles; D014867:Water; C009462:thioflavin T; D003903:Deuterium