Spectral Properties of Thioflavin T and Its Complexes with Amyloid Fibrils

Abstract

Comparative analysis of the absorption and fluorescence spectra and fluorescence excitation spectra of thioflavin T (ThT) in various solvents and in the composition of amyloid fibrils has shown that ThT, when excited in the region of the long-wavelength absorption band, fluoresces in the spectral region with a maximum at 478-484 nm. The appearance in aqueous and alcohol solutions of a fluorescence band with a maximum near 440 nm has been attributed to the presence in the composition of the ThT preparations of an impurity with an absorption band in the 340-350-nm range. The literature data showing that in glycerol ThT has a wide fluorescence spectrum with two maxima are due to the artifact connected with the use of a high concentration of the dye. It has been suggested that the cause of the low quantum yield of ThT aqueous and alcohol solutions is the breakage of the system of conjugated bonds due to the reorientation of the benzothiozole and benzaminic rings of ThT in the excited state with respect to one another. The main factor determining the high quantum yield of fluorescence of ThT incorporated in fibrils is the steric restriction of the rotation of the rings about one another under these conditions. The suggestions made have been verified by the quantumchemical calculation of the ThT molecule geometry in the ground and excited states.

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

“…5). This finding indicated that an increase in the ThT emission The observed emission enhancement phenomenon, induced by the endospore uptake, is consistent with the sensitivity of the photophysical properties of ThT to the viscosity of the environment: i.e., the emission quantum yield of ThT increases with the increase in the medium viscosity (72,74,81,82,88,90). Due to the single carbon-carbon bond between the two ring systems and the single carbon-nitrogen bond between the phenyl ring and the dimethyl amine (Fig.…”

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

“…The CR difference spectra, calculated by subtracting the spectra of CR alone and aggregate alone from those of the aggregates in the presence of CR, showed maxima at 540 nm (data not shown). The CR difference spectrum with a maximum at 540 nm is a characteristic of CR binding to the cross-b motif (40,41,44). We also observed that a change in the CR absorbance of the I2-state aggregate at 540 nm is larger than that of the I1-state aggregates, indicating that the former contains more cross-b motif.…”

“…CR binds with fibrillar aggregates and gives information about the cross-b motif (40,41,44). The CR difference spectra, calculated by subtracting the spectra of CR alone and aggregate alone from those of the aggregates in the presence of CR, showed maxima at 540 nm (data not shown).…”

Orthogonal High-Throughput Thermal Scanning Method for Rank Ordering Protein Formulations

“…A few of the most commonly used fluorescent dyes to probe protein unfolding include SYPRO Orange and 1-anilino-8-naphthalene sulfonate (6)(7)(8). Similarly, aggregate formation has been studied using thioflavin T (ThT), Nile Red, and Congo Red which have been known to bind specifically to amyloid-type fibrils (9)(10)(11)(12)(13). In the context of rapidly assessing the physical stability of proteins, a high-throughput approach using SYPRO Orange dye was previously described for examining unfolding propensity of protein mutants as well as rank-ordering protein formulations for their conformational stability (14).…”