Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages:Insights Obtained from Fluorescence Up-Conversionand Particle Size Distribution Measurements

International Journal of Molecular Sciences. 2020. Vol. 21. P. 6169.

Yu.G. Vainer, Rovnyagina N. R., Budylin G. S., Tikhonova T. N., Vasin S. L., Yakovlev A. A., Kompanets V. O., Chekalin S. V., Priezzhev A. V., Shirshin E. A.

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro.
However, the di erences in the time course of ThT fluorescence intensity and lifetime and other
physical parameters of the system, such as particle size distribution, raise questions about the correct
interpretation of the aggregation kinetics. In this work, we focused on the investigation of the
mechanisms, which underlay the di erence in sensitivity of ThT fluorescence intensity and lifetime to
the formation of protein aggregates during fibrillation by the example of insulin and during binding
to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using
dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond
resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as
much as a few percent of ThT bound to the high-anity sites that occur simultaneously with an
abrupt increase of the average particle size, particles concentration, and size heterogeneity. The
discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence
of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules
can only be detected using time-resolved fluorescence measurements in the sub-picosecond time
domain. The presence of a bound ThT subpopulation with similar photophysical properties was also
demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules
with a non-rigid microenvironment.

Keywords: фибриллы, time-correlated single photon counting (TCSPC), fluorescence up-conversion, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), fibrils, thioflavin T (ThT), коррелированный счет фотонов, флуоресцентная апконверсия, динамическое рассеяние света (ДРС), анализ траекторий наночастиц (АТН), тиофлавин Т