1-Anilino-8-naphthalenesulfonate: A fluorescent probe of membrane surface structure, composition and mobility

Summary

The binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalene-sulfonate (ANS⁻) on phospholipid vesicle membranes was studied to gain information about the structure and mobility in the polar head group region, and to determine the degree of mixing of lipids on the microscopic scale. The maximal degree of binding of ANS⁻ on dimyristoyl and dipalmitoyl-l-lecithin membranes is one ANS⁻ per four lecithin molecules, indicating a binding site composed of four polar head groups. ANS⁻ bound in this site has a long fluorescent lifetime (5 to 9 nsec) and high quantum yield (0.2 to 0.3), indicating that it is relatively inaccessible to the solvent water. The lack of paramagnetic quenching by added Mn²⁺ indicates that ANS⁻ bound to those fourmembered sites is also well shielded from added cations. Similarities in the temperature dependence of the binding constant and the reciprocal fluorescent lifetime indicate that the latter is determined by the propensity for polar head group motion and for water and ANS⁻ reorientation during the excited state of the molecule.

Membranes composed of lipids which lack a semi-polar head group (phosphatidic acid) or which have unfavorable polar head group conformations or strong interactions between the polar head groups (dimyristoyl ethanolamine) do not support the binding of ANS⁻ with a high quantum yield and long fluorescent lifetime. Incorporation of these lipids in a lecithin membrane decreases the maximal binding of ANS⁻ to a greater extent than can be explained on the basis of dilution of the lecithin with these lipids. A statistical model is presented, in which incorporation of one or more molecules of the second type into a four-membered lecithin binding site destroys the ability of this site to bind ANS⁻ with a long fluorescence lifetime. Agreement between this model and the results obtained with lipid mixtures indicate that egg phosphatidic acid and dimyristoyl ethanolamine mix well with dimyristoyl lecithin on the microscopic scale. The above criterion was also used to show that cholesterol mixes randomly with lecithin. In contrast to the behavior of the phospholipid mixtures, there was evidence for shortlived ANS⁻ species for cholesterol/lecithin mixtures with mole ratios between 0.6 and 1.0. This species is associated with binding sites in which the lecithin polar head groups are spaced out by the inclusion of cholesterol between the hydrocarbon chains of the lipid molecules. The effects of the ion carrier valinomycin and local anesthetics on the lecithin binding sites are discussed.