Fluorescent properties of thioflavin T incorporated in native and denatured proteins
The introduction gives a brief overview of the works on spectral properties of the fluorescent probe thioflavin T (ThT). The focus is on explaining the reason of the significant (by three orders) fluorescence quantum yield increase as a result of binding into ordered protein aggregates known as amyloid fibrils. Also unsolved problems are considered, one of them is the influence of blood plasma proteins (human serum albumin, HSA, and bovine serum albumin, BSA) on the intensity and fluorescence lifetime of ThT. The main content of this work are results of studies of spectra and fluorescence decay kinetics of ThT aqueous solutions in present of native and denatured proteins. It is shown that increasing of the protein concentration in the solution leads 84to 10-fold increase in the ThT fluorescence intensity. The fluorescence decay in this case is non-exponential and characterized by two-modal distribution. The increase in protein aggregation by adding ions (K + and Cs +) in solution and incubating the solution at 63 °C leads to a further increase of the emission intensity of the probe and increase of the contribution of the longer decay time mode. Based on the analysis of the data it was assumed that the change of spectral characteristics of the probe is a result of its incorporation into proteins. Two modes in the fluorescence decay correspond to the two ways of ThT incorporation into proteins. The longer decay time mode is formed by the fluorescence of the molecules that are incorporated into protein aggregates. ThT molecules incorporated into monomeric or aggregated macromolecules far away from their association contribute to a short decay time mode. This data suggests that the presence of proteins in solution in native denatured or partially aggregated forms leads to ThT fluorescence quantum yield increasing. The observed change is in one to two orders lower than in the case of presence of amyloid fibrils in solution, but for conducting quantitative content analysis of fibrils in vitro it is necessary to control the content of proteins in biological material. The quantum yield and fluorescence lifetime dependence on the proteins aggregation degree suggests that ThT is a sensitive probe for the investigation of the blood plasma protein aggregation.