Mapping the Path of Carbon

Now let us turn our attention to the reductive and synthetic reactions of photosynthesis. The basic carbon reduction cycle by which carbon dioxide is reduced to sugar phosphate involves at least twelve intermediate compounds. Some of these substances are found in very small concentrations. Many similar compounds are also present in the photosynthetic cell. In some cases they are closely linked by metabolism to the intermediates in the carbon reduction cycle. In order to understand the mechanism of photosynthetic carbon reduction, one must know the identity of the in- 

These problems resisted scientific investigation until the advent of modern biochemical methods. One of the most valuable of these new methods proved to be the use of radiocarbon as a tracer element to follow the newly incorporated carbon dioxide during photosynthesis in a green plant. Photosynthesizing plants do not discriminate significantly between ordinary carbon dioxide, C 02, and radioactive carbon dioxide, C 02. The plants incorporate C 02 into the intermediates in the carbon reduction pathway. 

Ruben et al. first used radiocarbon in studies of the path of carbon in photosynthesis (1940). They found that CO2 fixation in the dark is greater following preillumination. This is in accord with the concept that CO2 fixation occurs by dark reactions which use relatively stable chemical species formed in the light. They discovered that the radioactive product formed from labeled CO2 after preillumination was a carboxylic acid. These findings suggested that the path of carbon reduction in photosynthesis might very well include CO2 fixation mechanisms similar to those found in nonphotosynthetic plant tissues. 

Calvin and Benson and their co-workers used the radioisotope method to study carbon reduction in photosynthesis (Calvin and Benson, 1948, 1949). They allowed the plants to photosynthesize intermediate compounds from C 02 for short periods of time (a few seconds) and under a variety of experimental conditions. The plants were then killed, stopping the biochemical reactions. The radioactive products in the plant material were analyzed and identified. A careful study was made to determine the amounts of radiocarbon incorporated into chemical substances as a function of the experimental conditions. From the results of this study, they were able to map the path of carbon in photosynthesis. 

Comparison of the location of an unknown radioactive compound on the paper with this map gives a preliminary indication of the possible identity of the substance. One may then elute the radioactive compound from the paper chromatogram with water. Next, one mixes the labeled substance with an unlabeled sample of the suspected compound, and rechromatographs the two together. Following this cochromatography and radioautography, the paper 

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