368 Photosynthesis /20 : 4 



4. The Path of Carbon in Photosynthesis 



The pathways followed by the carbon of the C0 2 during its fixation and 

 conversion to hexose have been studied with radioactive carbon as a 

 tracer. To do this, C 14 2 is introduced into a suspension of photo- 

 synthesizing cells or isolated chloroplasts. If the reaction is stopped 

 after a brief time, one may determine from cellular extracts the com- 

 pounds containing labeled carbon. Many similar experiments may be 

 performed using different times of exposure to C 14 2 ; from these data 

 the relative amounts of the radioactivity in the various labeled com- 

 pounds may be plotted as a function of time. Thus, it is possible to 

 establish the order in which the compounds appear and hence, their 

 relationship to one another. 



In order to separate the various labeled compounds, a system called 

 paper radiochromatography is used. (The word chromatography is very 

 misleading because colors are not involved.) In paper chromatog- 

 raphy, the cellular extract is placed near one corner of a large square 

 of filter paper and dried. An edge is then immersed in a solvent 

 (phenol water) ; the various components at the origin migrate in a line. 

 The paper is dried, turned 90°, and the adjacent edge immersed in a 

 new solvent. The partly separated compounds migrate at different 

 rates in the two different solvents and so are arranged in a two-dimen- 

 sional array. The filter paper is dried. To find the radioactive, 

 labeled compounds, the filter paper is placed against a sheet of X-ray 

 film. The location of labeled compounds identifies them ; the intensity 

 of the darkening of the film shows the extent of labeling. 



Because the initial compounds form in a fraction of a minute, very 

 short exposures are necessary. Several of the steps known to occur 

 from these studies are summarized in Figure 4. Note that the final 

 compound formed is the hexose sugar, fructose-6-phosphate. Enzymes 

 exist within the chloroplast to change some of the fructose to glucose. 

 Some of the glucose in turn is polymerized to starch, whereas the 

 remainder is combined with fructose to form sucrose. This can be 

 represented by straight-chain formulas as shown in Figure 5. 



In order to confirm the scheme shown in Figures 4 and 5, it was 

 necessary to introduce the labeled C0 2 very rapidly into the mixture. 

 Then the entire process had to be stopped in a matter of seconds. It 

 was possible that the compounds found labeled might have reflected 

 the method used to stop the photosynthetic reaction. However, 

 plunging into boiling water, strong acid, and strong alkali all showed 

 3-phospho-glyceric acid as the first compound and confirmed the general 

 scheme shown. 



The initial reaction of C0 2 with ribulose-l,5-diphosphate is interesting 



