456 ANDRE T. JAGENDORF AND JOSEPH S. KAHN 



chance to accumulate under these circumstances. The Hght is then turned 

 off, stopping electron flow almost instantaneously. A very brief time later 

 ADP is added, allowed to incubate for 15 sec. in the dark, and then the 

 reaction mixture is killed. We find that some [^-P]-ATP has formed in the 

 dark, which is therefore a measure of the amount of X ~ ^^P carried in the 

 chloroplasts just after irradiation. The amount comes to i mju,mole per 

 1000 m/Ltmoles of chlorophyll in a remarkably reproducible fashion 

 (±20%). 



Although the experiment was simple in concept, scrupulous attention 

 had to be paid to the controls. Figure i shows that the chloroplasts were 

 pre-incubated for 8 minutes in the light with a large amount of cold 

 phosphate. This served to convert any internal ADP to unlabelled ATP. 



8min light 

 unlabelled P, 



i 



Dark, add ^^P| 



^ \ 



15 sec light Dark control 



/ \ / \ 



Dark, ADP 3% TCA Dark, ADP 3% TCA 

 15 sec I 15 sec I 



\ \ \ \ 



37oTCA ADP 3% TCA ADP 



I n m IV 



Fig. I. Protocol for "pre-loading" experiment, designed to show the existence 

 of a high-energy phosphate intermediate. 



If this were not done, internal ADP would combine with the added ^^p to 

 form large amounts of p-P]-ATP during the second illumination. 



Direct controls, as shown in Fig. i, included adding ^-P without a 

 second illumination (treatments III and IV), or killing the reaction prior 

 to addition of ADP in the dark (II and IV). 



Table I shows the results of one experiment. Treatment I, representing 

 illumination with ^-P followed by addition of ADP in the dark, formed 

 three times as much p-P]-ATP as any of the others. In other experiments 

 this ratio has varied from 1-5 to 6 (but the increment in p-P]-ATP due 

 to illumination remained the same in all experiments). Thanks to the 

 several controls we are sure that [^-P]-ATP was not formed in a dark 

 reaction, or in the light prior to the addition of ADP. 



By contrast with the successful results of this experiment no [^-P]-ATP 

 was ever found as a result of pre-illuminating with ADP and adding ^^P 

 in the dark afterwards. We therefore tend to conclude that a high-energy 

 ADP complex is probably not an intermediate here. 



