Hovor I. Fluorescent systems based on combinations of microenvironment-sensitive dyes for ratiometric determination of conformational changes of proteins

In this work the spectral-luminescent and photophysical properties of a number of squaraine, norsquaraine and styryl dyes in free state in solutions and when bound to various proteins were investigated. Regularities regarding the relationship between the structure and the spectral-luminescent and photophysical properties of dyes were established with the aim to create the ratiometric systems with Förster's resonance energy transfer (FRET) to develop a new approach for the determination of conformational changes in proteins. Squaraines are more sensitive to the polarity of the environment, what is manifested in a decrease in their quantum yield by 3.2-6.3 times during the transition from a solution in a nonpolar solvent (chloroform) to a polar medium (methanol), while the quantum yield of norsquaraines is almost independent from the polarity. The introduction of sulfo-groups increases the solubility of dyes in aqueous solution, but also it reduces the sensitivity of dyes to the polarity of environment. The presence of one carboxyl group also contributes to the solubility of the dyes in aqueous solution, but retains the sensitivity of the dyes to the polarity of the medium. Stable complexes of dyes with BSA are formed within some period of time, during which a gradual shift of fluorescence maximum of the spectrum is observed. The spectral properties of the complexes depend on the concentration of BSA. Norsquaraines are less sensitive to the presence of protein (BSA) than squaraine dyes. In contrast to dyes with sulfo-groups, which do not change their spectral properties in the presence of BSA, the spectral bands of squaraines are red-shifted by 10–36 nm relatively to the maxima in aqueous solutions upon formation of complexes with BSA.