PRINCIPLES OF TUACEK METHODOLOGY 41 



way by which caiboii dioxide is converted to carbohydrate or other 

 organic end products. Early work done with C" was limited by its 

 short half-life. However, the availal)ility of C'^ now allows detailed 

 experimentation. The procedure is generally to expose the experimental 

 material, usually algae or higher plants, to C'^02 for a predetermined time 

 under specific conditions and then to determine into what organic fraction 

 the labeled carbon has l)een incorporated. In addition to the conven- 

 tional methods of organic chemistry, paper chromatography (see Chap. 8 

 for details) has proved most valuable in this work, which requires the 

 detection and positive identification of the intermediate compounds. 



It was very soon shown with C'^ that algae in the dark were able to fix 

 about twenty times as much C()2 if they had been previously exposed to 

 light. Furthermore this fixation of CO2 in preilluminated plants was 

 shown to yield intermediates similar to those produced by reduction of 

 carbon during photosynthesis and different from those fixed in the dark 

 without preillumination. This and other evidence led to postulation of 

 two fairly distinct mechanisms, represented as follows: 



chlorophyll 



2H2O + light > 4[H] + O2 (1-36) 



CO2 + 4[H] ^ CH2O + H2O (1-37) 



Equation (1-36) represents the absorption of light by chlorophyll and the 

 transfer of energy to yield a photolysis of water, forming oxygen and 

 hydrogen, the latter in the form of a reducing agent. This reaction does 

 not seem to involve carbon or CO2 or the direct reduction of CO2. Efjua- 

 tion (1-37) represents the synthesis of organic intermediates from CO2, 

 the pathway of which may be traced with radiocarbon. 



Experimental observations indicated that C^^ was incorporated into 

 intermediates w^ithin a matter of seconds. At 10 sec after exposure of 

 Scenedesmus to C^Mabeled bicarbonate, radiocarbon was found in the 

 following compounds: phosphoglycerate, ribulose diphosphate, hexose 

 diphosphate, fructose phosphate, mannose phosphate, glucose phosphate, 

 sedoheptulose phosphate, ribose phosphate, ribulose phosphate, dihy- 

 droxyacetone phosphate, phosphoenol pyruvate. First, it may be noted 

 that nearly all the activity was found in phosphorylated compounds, 

 indicating the importance of phosphorus. Secondly, most of the C^^ was 

 found in the phosphoglyceric acid (see Chap. 2 for reference to experi- 

 ments showing that this was not due to an exchange reaction). Time 

 studies showed that the percentage of C^^ to be found in this compound 

 approached 100 at zero time. Thus phosphoglyceric acid was apparently 

 the first product of carbon dioxide reduction detectable under the exper- 

 imental conditions. Furthermore the other compounds found suggested 

 the formation of hexoses from phosphoglyceric acid by the reversible 

 reactions of glycolysis. 



