Revisiting the rational and proper use of di-8-ANEPPS fluorescent probe to study molecular interactions with lipid...

di-8-ANEPPS is a lipophilic fluorescent probe sensitive to membrane potential. It has been used for several purposes in biological imaging and physical chemistry spectroscopic studies. Among the latter, solute-lipid membrane interaction stands out in the past 30 years. Solute-induced perturbations of lipid membrane hydration result in spectral shifts of this probe, which are used to infer on how extensively solutes bind membranes. Inferring solute distribution to lipid membranes from lipophilic probes such as di-8-ANEPPS has the advantage of not requiring labelled solutes, but is limited by lack of a theoretical framework that relates experimental outputs to mechanistic molecular events. Hill plot-like data treatment is usually performed but the relationship remains empirical, based on chemical intuition rather than on a well-defined biophysical rationale. Here, we present a rationalization of the method that frames data analysis in order to attain precise molecular level conclusions based on the water-lipid partition constant and the lipid-to-solute saturation ratio. Experimental proof of concept is provided using peptide solutes to disturb lipid bilayers of unilamellar vesicles. The rational physical chemistry foundations of the methods are also addressed and used to improve the experimental protocol.