Salt-dependent changes of 9-aminoacridine fluorescence as a measure of charge densities of membrane surfaces.

1. When negatively-charged membranes or particles are added to a solution containing 9-aminoacridine and only low concentrations of salts, fluorescence from the dye molecules is decreased. The quenching mechanism is a result of an increase in concentration of the positively charged dye molecule at the surface (Searle, G.F.W. and Barber, J. (1978) Biochim. Biophys. Acta 502, 309-320). 2. Fluorescence quenching is released on adding salts, the effectiveness being generally dependent on the valency of the action used: C3+ > C2+ > C+, in line with a decrease in the surface potential. 3. The differential effect of cations is analyzed according to the Gouy-Chapman theory to obtain estimates for sigma, the net charge per unit area on a number of different surfaces. 4. It was found that in some cases the estimated value of sigma was not constant for a particular membrane system, but increased with salt concentration. The variation was much diminished, though not eliminated, when more rigid surfaces were examined. 5. An alternative method based on the distribution of a divalent cation (methyl viologen) in the diffuse part of the double layer was also used to estimate the overall charge density. This technique gave values lower than those obtained from 9-aminoacridine fluorescence changes. 6. It is argued that 9-aminoacridine cations distribute near localized, charged areas of surfaces, and that the salt-dependent estimates of sigma partly reflect charge redistribution accompanying changes in electrostatic screening by cations. It appears that 9-aminoacridine is a convenient probe to monitor changes in the heterogeneity of charged membranes.