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i 



CARBOXYLATION AND THE {CO2} COMPLEX 205 



Ruben suggested that the acceptor compound may be an aldehyde, and 

 and formulated the complex, {CO2}, as RCOCOOH. 



Attempts to identify the complex by coprecipitation with one of the 

 famihar plant acids (ascorbic, citric, fumaric, maleic, succinic, oxaUc, or 

 tartaric) gave no positive results. This appears understandable in the 

 light of the results obtained by the ultracentrifugation of the active 

 solution. According to Ruben, Kamen, and Perry (1940), the radioactive 

 product (obtained by the exposure of Chlorella for 20 minutes to C*02 in 

 the dark) has a sedimentation constant of 8.6 X lO-^^ or four times that 

 of sucrose, indicating a molecular weight of about 1000. Ruben and 

 Kamen (1940) have also measured the diffusion coefficient of the radio- 

 active complex, and found D = 0.44 X 10"^ cm.Vsec; from this they 

 estimated the molecular weight as being close to 1500. 



From all these experiments, Ruben and Kamen concluded that the 

 first step in photosynthesis is an enzymatic carboxylation of a colorless 

 molecule whose size and concentration is similar to that of chlorophyll. 

 We may add to this that Frenkel's experiments indicate that this car- 

 boxylation takes place outside the chloroplasts. 



Smith and Cowie (1941) also observed the absorption of radioactive 

 carbon dioxide by plants in the dark. They used sunflower leaves and 

 found that radioactive carbon dioxide can be used for the study of the 

 carbon dioxide-bicarbonate equilibrium, as well as that of the ICO2} 

 formation. Their results discussed on pages 192 to 195 have been 

 confirmed; and, as a new result. Smith and Cowie found that some 

 carbon dioxide is retained b}^ the leaves upon acidification (in agreement 

 with Ruben's observations on Chlorella); the acid-resistant complex 

 accounted for about 0.3 ml. carbon dioxide per 10 g. fresh leaves. This 

 absorption is almost entirely absent in frozen leaves, but freezing after 

 absorption does not destroy the complex. This indicates that freezing 

 affects the enzymatic system which catalyzes the carboxylation reaction. 

 The authors confirmed also the observation of Ruben and coworkers 

 that repeated evacuation and exposure to C*02 increases the amount of 

 absorbed activity (e. g., from 0.03 to 0.08 ml. in 1.3 g. leaves, after three 

 evacuations). The concentration of the acceptor in the leaves (assuming 

 a stoichiometric ratio of acceptor : carbon dioxide as 1:1) is 4 to 

 5 X 10~^ mole per liter, that is, it is similar to the concentrations of 

 chlorophyll in sunflower leaves (which is 3 to 5 times smaller than in 

 Chlorella cells). 



The radioactive complex, {C*02}, formed in the dark is used up 

 afterwards in light, thus confirming the assumption that its formation is 

 a preparatory step in photosynthesis. 



There is no reason why the formation of the complex, { CO2 } , should 

 not also be detectable by ordinary analytical methods. However, ob- 



