The H+/e ration in chloroplasts in 2. Possible errors in its determination.

The number of H+ released inside the chloroplast thylakoids per electron flowing to the terminal electron acceptor (the H+/e ratio) has been investigated using a pH indicator dye technique. The apparent H+/e ratio was measured by comparing the absorbance change due to H+-uptake outside the thylakoids in the absence of uncoupling agent, and the absorbance change due to the net H+-production associated with water-splitting after addition of uncoupling agent in the presence of ferricyanide. (1) The apparent H+/e ratio was found to be higher than 2, using methylamine as uncoupling agent (> 1 mM). It varied with the concentrations of added buffer. (2) The apparent H+/e ratio obtained with methylamine as uncoupling agent (with or without additional buffer) approached the value of 2 with decreasing concentrations of methylamine. (3) A consistent H+/e ratio of 2 was also obtained with gramicidin or the nigericin-type ionophore dianemycin over a relatively large concentration range, and did not vary with addition of extra buffer. (4) The buffering capacity of the chloroplast suspension was estimated and was found to be the same order of magnitude as that of methylamine at the concentrations used.