Probing biomembrane interfacial potential and pH profiles with a new type of float-like fluorophores positioned at varying distance from the membrane surface.

Fluorophores of a new type were synthesized to probe the electrostatic potential or pH profiles in the external interface of biomembranes. The probes consist of the pH-sensitive fluorophore 7-hydroxycoumarin, coupled to a tetradecyl (myristyl) tail by a spacer group of varying length. A positively charged group is included between the tetradecyl and spacer groups to encourage a float-like alignment in the membrane head-group region. Three probes of this type were compared with 4-heptadecyl-7-hydroxycoumarin the fluorophore of which is embedded in the lipid head-group domain. Thus, a ruler-type positioning of the fluorophores was obtained at about 0.2, 0.6, 1.0, and 1.3 nm from the surface. The membrane-bound probes were tested in well-defined liposomes prepared by extrusion with different surface charge densities and size. The predicted positioning of the float-like probes is supported by their binding behavior in liposomes and by steady-state and nanosecond time-resolved fluorescence anisotropy, as well as by their accessibility to different quenchers. The interfacial electrostatic potential (psi d) and pH (pHd) values were derived from the observed apparent pKa shifts of the probes. The obtained psi d and pHd profiles as function of the surface potential (psi 0) and distance from the membrane surface are in good harmony with predictions from nonlinear Gouy-Chapman theory. The electrokinetic potentials (zeta) of the liposome series, measured by Doppler-electrophoretic frequency shift of laser light scattering, are in good proportion to the probe data. When bound to yeast cells, these probes monitor interfacial changes in parallel with glucose-induced medium acidification.(ABSTRACT TRUNCATED AT 250 WORDS)