CHARACTERISTICS OF TRITIUM INCORPORATION INTO 

 ILLUMINATED CHLOROPLASTS 



Brlaji Colmaii and Wolf Vishniac 



It has been shown in previous experiments (1,2), that 

 chloroplasts, incubated with tritiim labeled water in the light, 

 incorporate tritium into chlorophyll a. More tritium is incorp- 

 orated in light than in the dark and little incorporation takes 

 place with boiled chloroplasts. The tritium label of the chloro 

 -phyll was found to be stable to acid but unstable to alkali, 

 which suggested that the chlorophyll was labeled at the C-10 

 position. 



Quantitative evaluation of the data was rendered difficult 

 by the unexplained loss of radioactivity from samples of purif- 

 ied chlorophyll a stored in organic solvents at -15°. This loss 

 has been found to be much more rapid when samples are dried prior 

 to counting in a windowless gas flow counter, and makes it impos- 

 sible to follow the kinetics of photo synthetic incorporation. In 

 order to minimize the loss of activity, samples were placed in 

 the counter while still in solution and allowed to dry in the 

 stream of counting gas. By this procedure the rate at which radio 

 -activity dissappeared from labeled chlorophyll could be followed. 

 Labeled chlorophyll a was obtained from chloroplasts which had 

 been illuminated in suspension together with tritiated water. The 

 chloroplasts were extracted with acetone, the acetone fraction 

 transferred to heptane, and chlorophyll a rapidly separated from 

 the other pigments by chromatography on thin layers of powdered 

 sugar. (Sucrose is applied to glass plates as a suspension in 

 methanol; the small amount of sucrose that dissolves in the meth 

 -anol serves to bind the remainder to the plate). Figure 1, curve 

 2, shows the loss of radioactivity from labeled chlorophyll a 

 isolated from chloroplasts. Curve 1. illustrates a similar decay, 

 but here the chlorophyll was labeled chemically. Purified chloro 

 -phyll a in ether was treated with tritiated water and 2% pyridine 

 at laboratory temperature. The enol-keto tautomerization resulting 

 from this treatment with base, presimjably leads to the replacement 

 of the protium on C-10 with tritivma. 



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