Fluorescent probes in biological systems are sensitive to environmental polarity by virtue of their response to the reaction field created by polarization of the dielectric medium. Classically, fluorophore solvatochromism is analyzed in terms of the Lippert equation and later variants, all of which rely upon the original reaction field of Onsager. A recent survey of the solvent dependence of EPR spin-label probes, which are responsive solely to the reaction field in the ground state without the complication of excited states, shows that the reaction field of Block and Walker performs best in describing the polarity dependence. In this model, the step-function transition to the bulk dielectric medium used by Onsager is replaced by a graded transition. Analysis of the Stokes shifts for representative fluorescent membrane probes, such as PRODAN, DANSYL, and anthroyl fatty acid, reveals that, of several different reaction fields (including that of Onsager), the Block-Walker model best describes the dependence on solvent dielectric constant and refractive index for the different probes simultaneously. This is after full allowance is made for all contributions involving polarizability of the fluorophore, a point that is frequently neglected or treated incorrectly in studies using biological fluorescent probes. By using the full range of polar and apolar solvents, it is then possible to establish a common reference for the polarity dependence of different fluorophores and to relate this also to the polarity dependence of biologically relevant spin-label EPR probes. An important application is calibration of the transmembrane polarity profile recorded by fluorescent probes in terms of the high-resolution profile obtained from site-specifically spin-labeled lipid chains. © 2009 by the Biophysical Society.
Marsh, D. (2009). Reaction fields in the environment of fluorescent probes: Polarity profiles in membranes. Biophysical Journal. Biophysical Society. https://doi.org/10.1016/j.bpj.2009.01.006