Local optical probing of proteins of various sizes and charges with FITC label

Fluorescein dyes are widely applied in fluorescence bioimaging to visualize a spatial distribution of substructures or to monitor a kinetics of certain processes in cells. However, optical properties of the dyes are sensitive to a number of physical and chemical parameters of the microenvironment and, when conjugated to a macromolecule, the dye can additionally serve as an indicator of these parameters in near-surface regions. The present study aims to reveal the relationships between the response of the fluorescein probe and the structure of the macromolecule to which it is attached. We conjugated fluorescein-5-isothiocyanate (FITC) to four proteins of different sizes and surface charges (hen egg-white lysozyme, bovine carbonic anhydrase II, bovine serum albumin, and luciferase from Photobacterium leiognathi) and analyzed the relationship of spectral, time-resolved, and polarization characteristics of the fluorescence probe with protein size and charge parameters. The study shows that ionic equilibrium of FITC and dielectric permittivity (ε) near the protein surface differ from those in the bulk phase at pH 6.50. For the first time, a strong negative correlation between local ε and the hydrophobic surface area of the protein and a strong positive correlation between net charge density of protein and the ratiometric fluorescence signal of FITC (I⁴⁸⁸/I⁴³⁵) were found. The combined effect of covalent and electrostatic binding of FITC to the protein was found to increase the rigidity of conjugation, allowing adequate estimation of protein size using the fluorescence depolarization technique.