Determination of DeltapH in Chloroplasts. 1. Distribution of [14C]Methylamine

Abstract

A method for the determination of ΔpH in chloroplasts which is based on the distribution of [14C]methylamine between the osmotic water and the medium was developed. It was found that [14C]sorbitol penetrates only the nonosmotic space and can be used to determine the osmotic space. [14C]Methylamine was found to be somewhat concentrated in the chloroplasts in the dark but was extensively concentrated in the light, indicating a ΔpH of more than 2 units. Electrontransport cofactors increased this gradient while uncouplers decreased it. The maximal ΔpH was found when the medium pH was around 8.5, thus agreeing with the optimal pH for phosphorylation and electron transport. Comparison of the pH-dependence and the light-intensity-dependence of the extent of proton uptake and ΔpH indicates that the extent of proton uptake is essentially a measure of the chloroplasts buffering power but not of the ΔpH. The membrane potential across the chloroplast membrane was determined from the distribution of 36Cl and 86Rb and was found to be negligibly small in the light.