A QUANTITATIVE MODEL FOR USING ACRIDINE-ORANGE AS A TRANSMEMBRANE PH GRADIENT PROBE

Monitoring the acidification of the internal space of membrane vesicle s by proton pumps can be achieved easily with optical probes. Transmem brane pH gradients cause a blue-shift in the absorbance spectrum and t he quenching of the fluorescence of the cationic dye acridine orange. It has been postulated that these changes are caused by accumulation a nd aggregation of the dye inside the vesicles. We tested this hypothes is using liposomes with transmembrane concentration gradients of ammon ium sulfate as model system. Fluorescence intensity of acridine orange solutions incubated with liposomes was affected by magnitude of the g radient, volume trapped by vesicles, and temperature. These experiment al data were compared 60 a theoretical model describing the accumulati on of acridine orange monomers in the vesicles according to the inside -to-outside ratio of proton concentrations, and the intravesicular for mation of sandwich-like piles of acridine orange cations. This theoret ical model predicted quantitatively the relationship between the trans membrane pH gradients and spectral changes of acridine orange. Therefo re, adequate characterization of aggregation of dye in the lumen of bi ological vesicles provides the theoretical basis for using acridine or ange as an optical probe to quantify transmembrane PH gradients. (C) 1 998 Academic Press.