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Measurements of transmembrane pH differences of low extents in bacterial chromatophores

A study with the fluorescent probe 9-amino, 6-chloro, 2-methoxyacridine

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Abstract

In chromatophores from photosynthetic bacteria the interaction of the fluorescent monoamine, 9-amino, 6-chloro, 2-methoxyacridine (ACMA), with the membrane is evaluated and described by an S-shaped adsorption isotherm. This phenomenon is hysteretic, as indicated by the difference between the adsorption and desorption branches of the binding isotherm. Maximal saturation of adsorption is reached at one ACMA per one to four lipid molecules, indicating that the probe binds in its neutral form. Adsorption of the probe on the membrane causes a large quenching of its fluorescence, which is explaind as being due to hypochromic effects following stacking and aggregation in a medium of low dielectric constant. A further quenching of fluorescence is brought about by imposing artificially induced transmembrane ΔpH's. This latter phenomenon titrates in at increasing ΔpH values and approaches saturation when ΔpH is ≧2. The dependence of ΔpH on the observed quenching of fluorescence is predicted by considering a model based on the equilibrium distribution of the amine between two phases at different pH's, in which adsorption of the probe on the membrane is used to evaluate its free concentration in the inner and outer compartments of the chromatophore vesicle. It is proposed that the equation thus obtained should be used to measure ΔpH from the quenching of ACMA fluorescence.

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Abbreviations

ΔpH:

transmembrane pH difference between the inner and outer compartments

Q:

quenching of fluorescence

BChl:

Bacteriochlorophyll

ACMA:

9-amino-6-chloro-2-methoxyacridine

9AA:

9-aminoacridine

Tricine:

N-tris-(hydroxymethyl)methylglycine

MES:

2-morpholinoethanesulfonic acid

FCCP:

carbonylcyanide-p-trifluoro-methoxy-phenylhydrazone

CCCP:

carbonylcyanide-m-chloro-phenylhydrazone

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  1. Section of Biophysics, Department of Biology, University of Bologna, I-40126, Bologna, Italy

    R. Casadio

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  1. R. Casadio
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Casadio, R. Measurements of transmembrane pH differences of low extents in bacterial chromatophores. Eur Biophys J 19, 189–201 (1991). https://doi.org/10.1007/BF00196345

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  • DOI: https://doi.org/10.1007/BF00196345

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