Kinetics of acid efflux from chloroplasts following the acid-base transition

Swollen chloroplasts, suspended in succinic or other acids at pH 4 were mixed with alkali in a stopped-flow apparatus to permit observation of the pH of the medium following an acid-base transition. Immediately after mixing the observed pH values were as much as 0.6 units more alkaline than the final equilibrium pH, then dropped to the equilibrium value over a period of several seconds. Experimental results support the idea that most of the slowly appearing acidity represented diffusional loss from the thylakoids of organic acid molecules, previously absorbed from the initial acid medium. Time-courses for the approach to equilibrium pH showed apparent first-order kinetics, with two distinct components: (a) the major portion of acid loss, having a Q 10 of 2, and (b) a minor residual portion of the acid loss, having a Q 10 of 1.2. Component (a) could be distinguished by faster kinetics only at 20° or above. Uncouplers had relatively little effect on (b), but diminished the extent and duration by accelerating the velocity of initial acid loss (a). The combined results of several diverse experiments showed a correlation between the ability to make ATP, and the existence of large and relatively slow initial acid loss (a). Kinetics of this initial phase are similar to those of the post illumination pH drop studied previously, under equivalent conditions. The very slow response times of the glass pH electrode in unbuffered solutions described previously was found here to limit the observed rate constants for the chloroplast light-induced pH rise, thereby calling into question the accuracy of some previously published kinetic studies.