1634 CHEMICAL PATH OF CARBON DIOXIDE REDUCTION CHAP. 36 



compounds which are present in relatively small amounts could be due to 

 the reversal of respirative (or fermentative) decarboxylations, and have no 

 relation to photosynthesis. 



The existence of these complications was not known when the first car- 

 bon tracer studies of photosynthesis were made by Ruben and co-workers 

 with the short-lived carbon isotope, C(ll). It is therefore difficult to de- 

 cide which of their observations (described in Vol. I, pages 201 and 241) 

 remain significant from the point of view of the mechanism of photosynthe- 

 sis. 



Ruben, Kamen and co-workers found (1939, 1940) that in the dark, C"02 was 

 incorporated, by an enzymatic reaction, into a water-soluble molecule, with a molecular 

 weight of about 1000, forming a carboxyl group. After brief illumination, they found 

 most of the active carbon in a similar (perhaps, the same) large molecule, but by now 

 not, or not exclusively, in a carboxyl group. The active material was, however, still 

 soluble in water and precipitable by barium. It was later observed, with some surprise, 

 by Frenkel (1941) (also at Berkeley) that in the dark, the C* uptake was localized in 

 the cytoplasm. After brief illumination, however, the radioactivity was found pre- 

 dominantly in the chloroplasts. 



The amount of C(ll) taken up after several hours of exposure to C*02 in the dark 

 was about 0.05 mole/liter of cell volume — roughlj^ equivalent to the amount of chloro- 

 phyll present; however, the tagged product was not bound to chlorophyll. (We noted 

 above that it could be extracted with water.) 



It seemed natural at that time to identify the C*02 acceptor compound, whose for- 

 mation in the dark was indicated by these experiments, with the often postulated sub- 

 strate of photochemical reduction (which we have variously symbolized by ICOal, 

 ACO2 or RCOOH). However, this identification presented certain difficulties. One of 

 them was the slowness with which C(ll) was taken up: about an hour was needed to 

 "saturate" the cells with radiocarbon in the dark (c/. fig. 21, Vol. I); the same amount 

 of carbon can be absorbed in about 20 seconds, either by photosynthesis in strong Hght, 

 or by "pick-up" after intense photosynthesis in the dark (Vol. I, p. 206). 



Two explanations of the slowness of the observed C* uptake could be suggested. 

 One is that this uptake is slow when it has to proceed through isotopic exchange: 



(36.3) C*02 + RCOOH , CO2 + RC*OOH 

 but becomes /asi when it can occur by C*02 addition to free acceptor: 



(36.4) C*02 + RH , RC*OOH 



(However, on page 203, the "one hoxir uptake" itself was tentatively attributed to 

 reaction with free acceptor — -in order to be able to explain the occurrence of additional 

 CO2 uptake after evacuation.) 



During intense photosynthesis, in a medium which is low in carbon dioxide, the ac- 

 ceptor may be present in the photostationary state predominantly in the decarboxylated 

 state, RH. When illumination is stopped, CO2 rushes in, in a rapid gulp, as observed 

 in "pick-up" experiments. In the experiments of Ruben and Kamen, on the other hand, 

 the acceptor could have been present mainly in the carboxylated form, RCOOH, and 

 C(ll) could enter it only by the slow exchange reaction (36.3). 



A second possible reason for the observed slowness of the C * uptake in the dark has 

 become apparent after it had been established that C*02 can be taken up by mechanisms 



