Light-Induced pH Gradients in Isolated Spinach Chloroplasts

Abstract

The fluorescence quenching of certain fluorescent, weakly ionizing amines has been used to estimate the pH difference (ΔpH) between the intrathylakoid spaces of chloroplasts and the external medium under various conditions. The amines N-(1-naphthyl)ethylenediamine and 9-aminoacridine gave consistent and similar estimates of ΔpH provided the concentration ranges 0.25 - 2 μM and 0.05 - 0.5 μM, respectively, were not exceeded. Values for ΔpH of up to 4 units were estimated for control chloroplasts. Only a small decrease in ΔpH was caused by FCCP and DCMU concentrations that decreased photophosphorylation rates to 10-20% of control. The kinetics of the onset and decay of ΔpH, and of the uptake and release of protons by the chloroplasts, were followed after the sudden application or withdrawal of actinic light. These kinetics suggest a model for the ionic relations of stripped chloroplasts with internal buffering capacity, [α], and dissociation constant, K_a, as parameters. At pH8, [α] is about 80 mol m^-3 and K_a is 0.07 mol m^-3 (pK_a = 4.15). The model is successful in predicting the relation between [H⁺]_I (the internal proton concentration) and ΔH⁺_o (the light-driven uptake of protons from the external medium), as well as the size of ΔH⁺_o , but needs extending to deal with published experiments on the light-driven translocation of chloride ions.