The quenching of an intramembrane fluorescent probe. A method to study the binding and permeation of phloretin through bilayers

Phloretin and phloretin-like dipolar non-electrolytes strongly quench the fluorescence of several membrane-bound probes, including 1,6-diphenylhexa-1,3,5-triene and anthroyl derivatives of long-chain fatty acids. Fluorescence intensity measurements therefore provide a simple and sensitive method to study the equilibrium binding properties and permeability of phloretin-like molecules in biological and artificial membrane systems. The dissociation constants for the binding of phloretin and naringenin to phosphatidylcholine vesicle membranes are determined, assuming the Stern-Volmer relation, from the fluorescence intensity of intramembrane probes as a function of phloretin and naringenin concentrations. Results (phloretin, $\text{9 ± 1}\text{μM}$; naringenin, $\text{21 ± 4}\text{μM}$) agree with the dissociation constants obtained using absorption titration performed in the absence of fluorescent probes. Fluorescence nanosecond lifetime measurements show that the mechanism of quenching of diphenylhexatriene and 16-anthroylpalmitic acid by phloretin and naringenin is largely diffusional in nature. The transmembrane movement of phloretin through phosphatidylcholine vesicles was observed by the stopped-flow technique, in which phloretin is mixed rapidly with a vesicle solution containing a membrane-bound fluorescent probe. The time course obtained by fluorescence measurements was identical to that obtained in a parallel measurement of the time course of optical absorption of phloretin. Stopped-flow data for the permeability of phosphatidylcholine liposomes and red blood cell membranes are also presented. The use of a membrane-bound indicator greatly extends the range of concentrations and pH values as well as the types of systems which can be characterized by optical means.