Calibration of the response of 9-amino acridine fluorescence to transmembrane pH differences in bacterial chromatophores

Abstract

The spectral characteristics of absorption and fluorescence emission of 9-amino acridine are not altered by the interaction with bacterial chromatophores, except for the attenuation of both the absorption and emission following the formation of a protonic gradient. The lifetime of fluorescence of the dye is significantly affected in the presence of membranes, and even more following illumination. The shortening of the lifetime induced by light is reversible and prevented by nigericin and K +. The onset kinetics of the fluorescence quenching following the generation of an artificial transmembrane pH difference is temperature dependent, with an activation energy of 17 ± 3 kcal/mol. The effect of pH on the rate constants is consistent with a model assuming that the diffusion of the unprotonated species is the limiting step in the quenching phenomenon. The response of 9-amino acridine to artificially imposed ΔpH's has been utilized as a calibration method for the measurements of the light-induced protonic gradient. The apparent inner volume of chromatophores, evaluated from the extraplation of the response at ΔpH = 0, was found to be much larger (15- to 40-fold) than the true osmotic volume, indicating that most of the dye is bound to the membrane when accumulated into the inner lumen.