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

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences 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 difference 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-affinity 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.

Dissection of the deep-blue autofluorescence changes accompanying amyloid fibrillation.

Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (∼350 nm excitation; ∼450 nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.

Grading cartilage damage with diffuse reflectance spectroscopy: Optical markers and mechanical properties.

Osteoarthritis (OA) is one of the most common joint diseases worldwide. Unfortunately, clinical methods lack the ability to detect OA in the early stages. Timely detection of the knee joint degradation at the level of tissue changes can prevent its progressive damage. Here, diffuse reflectance spectroscopy (DRS) in the NIR range was used to obtain optical markers of the cartilage damage grades and to assess its mechanical properties. It was observed that the water content obtained by DRS strongly correlates with the cartilage thickness (R = .82) and viscoelastic relaxation time (R = .7). Moreover, the spectral parameters, including water content (OH-band), protein content (CH-band), and scattering parameters allowed for discrimination between the cartilage damage grades (10-4 < P ≤ 10-3 ). The developed approach may become a valuable addition to arthroscopy, helping to identify lesions at the microscopic level in the early stages of OA and complement the surgical analysis.

Binding of thioflavin T by albumins: An underestimated role of protein oligomeric heterogeneity.

Amyloid fibrils formation is the well-known hallmark of various neurodegenerative diseases. Thioflavin T (ThT)-based fluorescence assays are widely used to detect and characterize fibrils, however, if performed in bioliquids, the analysis can be biased due to the presence of other, especially abundant, proteins. Particularly, it is known that albumin may bind ThT, although the binding mechanism remains debatable. Here the role of low-order albumin oligomers in ThT binding is investigated using time-resolved fluorometry and size-exclusion chromatography. Under conditions used, the fraction of dimers in human (HSA) and bovine (BSA) serum albumin solutions is as low as ∼7%, however, it is responsible for ∼50% of ThT binding. For both albumins, the binding affinity was estimated to be ∼200 and ∼40µM for monomeric and dimeric species, respectively. Molecular docking suggested that ThT preferentially binds in the hydrophobic pocket of subdomain IB of albumin monomer in a similar position but with a variable torsion angle, resulting in a lower fluorescence enhancement (∼40-fold) compared to amyloid fibrils (∼1000-fold). Dimerization of albumin presumably creates an extra binding site at the subunit interface. These results demonstrate the underestimated role of low-order albumin oligomers that can be highly relevant when analyzing drugs binding using fluorescence spectroscopy.