Adsorption of Amines to Thylakoid Surfaces and Estimations of ΔpH

Abstract

The adsorption of the fluorescent, weak amine ΔpH indicators 9-aminoacridine and N-(1-naphthyl)ethylenediamine to thylakoids of pea chloroplasts in the dark was measured as a function of amine concentration, at several pH's between 8 and 5, and with varied MgCl₂ concentration. It was concluded that it was the protonated form of the amine that was adsorbed. Maximum amounts adsorbed, saturation occurring at about 1 mM amine concentration in the presence of 5 mM KCl and 10 mM MgCl₂, were up to 1 mol (mol Chl)^-1. This was calculated as exceeding the number of available surface negative charges (0.26 for a charge density of 0.025 C m^-2). An apparent binding constant of 400-500 μM (concentration for half saturation) was noted for both amines under the above conditions.

A model was developed that enabled the estimation of the relative amounts of amine bound to the inside and outside thylakoid surfaces in the steady state, in the light. By this means corrections to the apparent ΔpH calculated from fluorescence quenching of the amines could be established. These corrections amounted to about 1 pH unit for the diamine and more for 9-aminoacridine. The predictions of the model were in good agreement with experimental relations between estimated, light-induced ΔpH values and amine concentration.

The implications of a lower ΔpH for earlier estimations of proton motive force in relation to photophosphorylation are briefly considered.