QUANTITATIVE ESTIMATION OF THE PHOTOSYNTHETIC PROTON BINDING INSIDE THE THYLAKOIDS BY CORRELATING INTERNAL ACIDIFICATION TO EXTERNAL ALKALINIZATION AND TO OXYGEN EVOLUTION IN CHLOROPLASTS

The external alkalinisation ΔpHe, or the rate of oxygen evolution υO2, of a suspension of envelope-free chloroplasts was correlated with their internal acidification, estimated from the transmembrane ΔpHei. Knowing the external buffer value, the concentration of the total protons moved Hi was calculated from the ΔpHe, measured with a glass electrode ([Hi] was also obtained from υO2), and the free proton concentration [H+i] was determined from ΔpHei, measured with 9-aminoacridine. This gives a ratio γi = ∂ [Hi] ∂ [H+i], which is independent of the thylakoids internal volume. Within a large pHi range, scanned by varying the light intensity, γi was kept reasonably constant; it was hardly sensitive to pHe. This apparent invariability implies a continuous change of the internal buffer value βi with pHi, since βi γi = -2.3... 10-pH, a relationship which includes neither the total concentration of protonizable groups [Ai] nor pKi. As γi ≈ Ki[Ai] (Ki + [H+i])2, to keep γi constant when pHi drops, pKi and [Ai] must increase. This may be achieved by a progressive unmasking of anionic functions, initially inaccessible in the membrane. The relative slowness of this process may explain why γi calculated from the initial kinetics was sometimes smaller in high than in low light, where it always equalled that measured from the steady-state amplitude at all intensities. A small deficit of [H+i] deduced from what could have been expected from ΔpHe may reflect a limited binding of protons in the membrane itself, about 1 H+ for 30-130 chlorophylls (γi could be between 70 and 240, more frequently around 100); these numbers varied depending on the samples, but were constant for a given preparation. © 1979.