The H+/e Ratio in Chloroplasts is 2. Possible Errors in its Determination

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Abstract
The number of H+ released inside the chloroplast [spinach] thylakoids per electron flowing to the terminal electron acceptor (the H+/e ratio) was 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. 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. 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. 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. 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. Possibly the high H+/e ratios reported by Fowler and Kok reflect a failure to take account of the change in buffering capacity of the chloroplast suspension on adding uncoupling concentrations of methylamine. Other possible sources of error in the estimation of H+/e ratio by different techniques are also discussed.

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