Effect of Uncouplers on the Light-induced Ph Rise with Spinach Chloroplasts.

A readily reversible rise in pH of the medium occurs when unbuffered spinach chloroplasts are illuminated, especially when the initial pH during incubation in the dark is at or below 6.5 (1, 2). This rise in pH had been predicted from an examination of the chemiosmotic mechanisms for phosphorylation detailed by Mitchell (3). It was correlated, in at least a preliminary way, with the existence in the same chloroplasts of a light-induced high energy (nonphosphorylated) state designated X,, a possible intermediate in the complete photophosphorylation mechanism (1, 4, 5). It is likely that the pH rise is closely related to a light-driven efflux of potassium ions recently described by Dilley (6). The maximum extent of the pH rise seems to be determined by an equilibrium between two opposing reactions: light-dependent formation and a light-independent decay. The latter can be measured directly during a postillumination period. It is of interest to see whether inhibitory reagents affect the yield by depressing the rate of formation or by increasing the rate of the back reaction. Addition of the inhibitor in a postillumination period gives conclusive evidence as to whether decay is affected or not, but the kinetics of formation in itself provides only indirect evidence as to whether the true formation rate is affected or not. It was found previously (2) that the initial rate of the pH rise was too rapid for quantitative determination by means of the pa-stat. It will be shown here that the direct recording of the pH can be used for this purpose, with a fair degree of reliability. Within the limitations of the techniques, the various uncouplers have been analyzed as to their mode of action.