204 FIXATION OF CARBON DIOXIDE CHAP. 8 



The concentration of the complex, {CO2}, is of the same order of 

 magnitude as that of chlorophyll. (Chlorophyll constitutes as much as 

 5% of the dry weight of Chlorella, cf. page 411, corresponding to an 

 average concentration of about 0.01 mole per liter.) However, the 

 acceptor is not chlorophyll, for the aqueous cell extract (which contains 

 all radioactive carbon) is colorless. Furthermore, cells with different 

 chlorophyll concentrations show no differences in C*02-absorbing ca- 

 pacity, and etiolated plants also are able to absorb carbon dioxide. 



Frenkel (1941) exposed Nitella plants to C*02 for 25 minutes, and 

 disintegrated the cells in 0.5 Tkf glucose solution (Nitella cells disintegrate 

 without grinding). He separated the cell sap and the cytoplasm from 

 the (mostly intact) chloroplasts by centrifuging, and tested the activity 

 of different fractions (after preliminary boiling with 12 iV hydrochloric 

 acid for the removal of carbonates). Surprisingly, no activity was 

 found in the chloroplasts; 90% of the total activity was found in the 

 colorless supernatant solution. When similar plants were exposed to 

 radioactive carbon dioxide in light, four-fifths of the absorbed activity 

 were found in the chloroplast fraction, and the active substance could not 

 be extracted by a 0.5 M glucose solution. Nitella cells, crushed before 

 exposure to radioactive carbon dioxide, formed none of the acid-resistant 

 carbon dioxide complex whether they were exposed in dark or in light. 

 When intact cells were first exposed to C*02 in the dark and then crushed 

 and exposed to light, no transfer of activity from the aqueous phase 

 into the chloroplasts could be observed. 



Thus, it appears that the carbon dioxide acceptor is either only 

 loosely bound to the chloroplasts (so that it can be removed by a short 

 contact with glucose solution) or, more probably, is not contained in the 

 chloroplasts at all, but rather in the cytoplasm or in the cell sap. Further- 

 more, intact cells seem to be a prerequisite both for the formation of 

 {CO2} and for its reduction in light. 



The following information was obtained by Ruben and coworkers as 

 to the chemical nature of the carbon dioxide-acceptor complex in Chlorella 

 cells. Over 70% of the active substance is precipitated by barium ions 

 in 80% ethanol, and 30-50% of the active barium salt is transformed 

 into carbonate by dry distillation. The theoretical value for complete 

 decarboxylation of a barium salt of a carboxylic acid, with C* located 

 in the carboxyl group, is 50%. It thus seems that most, if not all, of 

 the carbon dioxide in the {CO2} complex is contained in a carboxyl 

 group. In addition to the carboxyl group, the complex apparently 

 contains one or several hydroxyl groups, whose presence was indicated by 

 the Schotte-Baumann test (esterification with benzoyl chloride and 

 extraction of the active esters with chloroform). More recently (1943), 



I 



