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Articles

Use of Uncoupling Acridine Dyes as Stoichiometric Energy Probes in Chloroplasts ¹

^a Johnson Research Foundation, Department of Biophysics and Physical Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104

In a suspension of spinach chloroplasts the fluorescence of atebrin and other uncoupling acridine dyes is quenched upon energization which is associated with a proportional binding of the dyes to the organelles. There is a stoichiometric relation between the amount of dye bound and the actual steady state level of energy. When the concentration of atebrin is increased in energized chloroplasts the fluorescence is completely quenched until a certain concentration is attained above which the response sharply declines. Such titrations with atebrin were carried out under conditions of partial electron transport governed by photosystems I and II, in the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and cyanide, respectively, and of complete electron transport governed by the two photo-systems. The sum of the saturating amounts of atebrin obtained in these partial electron flow systems equals that obtained in the complete system. This lends strong support to the view that two sites of energy conservation are coupled to the linear photosynthetic electron transport.

When ATP was the energy donor the saturating amounts of atebrin were the same in both control and cyanide-treated chloroplasts, indicating that the energy-conserving mechanism was unimpaired in the latter.

Removal of the chloroplast-coupling factor by ethylenedia-minetetraacetate treatment leads to inhibition of the probe responses, which can be restored again in recoupled chloroplasts.

³ On leave from the Department of Botany and Plant Pathology, Michigan State University, East Lansing, Mich. 48823.

¹ This investigation was supported in part by a United States Public Health Service International Postdoctoral Research Fellowship No. 1-F05-TW-1821-01, and a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) to R.K., and a grant from the National Science Foundation No. GB 22657 to S.I.