214 



Brian Colman and Wolf Vishniac 



Chlorophyll a treated with tritiated water under neutral 

 conditions, that is in boiling ether, incorporates only a fraction 

 of the activity of that treated under basic conditions and the 

 resultant label is relatively stable (Fig. 2, curve 2, cf. curve 1.)- 

 Phaeophytin a labeled by each of the two procedures shows the same 

 types of decay as chlorophyll a. During the loss of radioactivity 

 there is no chemical change in the chlorophyll or phaeophytin as 

 judged by spectroscopic observation. Allomerized chlorophyll a, 

 prepared by the oxidation of chlorophyll a in methanol with gaseous 

 oxygen, was treated with tritiated water in 2fo pyridine in ether. 

 The small amount of radioactivity incorporated and its stability 

 (Fig. 2, curve 3) are consistent with the notion that the tritium 

 label of the intact chlorophyll a is located on C-10. Heating 

 allomerized chlorophyll a in ether in the presence of TOH did not 

 lead to significant labeling either. 



The loss of radioactivity from chlorophyll does not reflect 

 evaporation of tritiated water originally introduced into the 

 reaction mixture. The procedure for the isolation of the chloro- 

 phyll precludes the retention of any such water. Instead the 

 disappearance of tritium suggests that it is either given off as 

 water vapor or as hydrogen gas resulting from an unknown exchange 

 reaction. 



The light-dependant incorporation of tritiim into chloroplast 

 components is affected by CMU (p-chlorophenyl-l,l-dimethyl urea). 

 CMJ has been reported (3,i+) to inhibit the Hill reaction, the 

 reduction of TPN and photo synthetic phosphorylation in chloroplast 

 preparations. Pea chloroplasts were incubated with tritiated water 

 under the conditions described in Table 1. At the end of the 

 experiment, the chloroplasts were extracted with acetone and the 

 acetone fraction transferred to heptane. The residue of the chloro 

 -plasts was then succesively extracted with isooctane, methanol 

 and water. The data in Table 1. confirm the earlier results that 

 more activity is taken up by the component pigments of the heptajie 

 fraction in the light than in the dark, and also show that this 

 light stimulated uptake is inhibited by CMU. 



The accximulation of tritium in the isooctane fraction in the 

 presence of CMU is interesting. This fraction contains at least 

 two quinones which incorporate tritium from water in the light. 

 While tritium incorporated into a quinone would be expected to be 

 readily exchangeable with water, it had been suggested to us by 

 Dr. Calvin that the retained activity could result from the 

 rearrangement of a suitable reduced quinone, for example, reduced 

 Co Q, to form a chroman ring with the concomitant migration of 



