INDOPHENOL DYES: CATALYSTS AND UNCOUPLERS 

 OF PHOTOPHOSPHORYLATION 



Donald L. Kelster 



The indophenol dyes, especially 2 ,6-dlchloroindophenol (DCI) 

 and 2,3' ,6-trlchloroindophenol (TCI) have been known to be .^. 

 excellent oxidants for measuring the Hill reaction since ig'^S^ '> 

 In 195'iC2), the demonstration was made that isolated chloroplasts 

 under the influence of light could phosphorylate ADP and in 

 1958'35 it was discovered that the reduction of TPN was accom- 

 panied by ATP formation. Subsequently it has been shown that ATP 

 formation was coupled to the reduction of other Hill oxidants 

 including ferricyanide , FMN and cytochrome c. However, it has 

 been reported in the literature that the reduction of the iodo- 

 phenol dyes was not accompanied by photophosphorylation^ »^ K 

 This view has led to the postulation that these compounds were 

 reduced by some component of the electron transport chain prior 

 to the site of phosphorylation. 



Earlier this year three widely separated groups, Shen et gj..^°^ 

 in China, Gromet-Elhanan and Avron^'^ in Israel, and our own^^^ , 

 independently discovered that phosphorylation was coupled to the 

 reduction of indophenols. These compounds are potent uncouplers 

 of photophosphorylation in their oxidized form and therein lies 

 the reason that the phosphorylation coupled to their reduction 

 was previously overlooked, 



METHODS 



Unless otherwise specified each reaction mixture per 3 ml 

 contained the following in umoles: Tris, 150; ADP, 2; MgClp, 5; 

 ^^Pi, 2-10; and chlorophyll as noted. After illumination the 

 reactions were terminated by the addition of 0.3 ml of 50% 

 trichloroacetic acid (TCA) and uptake of 32pi determined as 

 previously noted^°\ Anaerobic experiments were performed in 

 Warburg flasks or in 50 ml Erlenmeyer flasks fitted for flushing 

 with gas. The flasks were flushed with a strong stream of argon 

 and shaken throughout the experiment. Ten minutes of flushing 

 was found sufficient to remove essentially all the oxygen. Where 

 reduced DCI or TCI was added, it was reduced with hydrogen using 

 platinum asbestos as catalyst thus eliminating any other reducing 

 agents. One drop of bromine water was added to samples for J'^Pi 

 uptake determinations after the addition of TCA to oxidize the 

 reduced indophenols and prevent their interference with the 

 determination. Cytochrome c was the Type III preparation of the 

 Sigma Chemical Company. 



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