The behavior of 9-aminoacridine as an indicator of transmembrane pH difference in liposomes of natural bacterial phospholipids

Abstract

The behavior of 9-aminoacridine as an indicator of pH differences artificially set across a membrane has been reexamined in liposomes prepared from bacterial phospholipids extracted from chromatophores of Rhodopseudomonas capsulata grown photoheterotrophically. The dye behaves as an ideal indicator for pH differences lower than about three units; at higher pH's the expected linear dependence of Q/(100-Q) vs. pH is no longer strictly observed. Similarly a linear dependence upon the volume of the liposomes added has been verified. The amine ceases to respond to pH changes when the pH of the external medium exceeds the value of 10, corresponsing to the pKa of 9-aminoacridine. The apparent volume of the inner phase of liposomes, as calculated from fluorescence quenching, but not the slope of dependence of fluorescence on pH, appears to be affected by several factors, including the ionic composition, the osmolarity of the external medium, and the microscopic structure of the liposomes. Millimolar concentrations of earth-alkaline cations diminish the apparent internal volume of liposomes, in agreement with the complexing effect of these ions on phospholipid bilayers. The osmotic response of the apparent inner volume has also been verified; this parameter decreases linearly with the reciprocal of the external osmolarity, as expected from the van't Hoff relation; an osmolarity exceeding 0.3 M is, however, necessary in order to observe this effect.