REACTIONS OF CARBON DIOXIDE IO7 



became concentrated; nevertheless they found much of the 

 tracer in the Hght was fixed in the carboxyl groups of a-keto 

 acids. Subsequently the long-lived tracer ^'*C became avail- 

 able and was exploited in the researches of Calvin and col- 

 leagues (1949, 1 951) and Gaffron and colleagues. In many 

 of the experiments a steady state of photosynthesis was 

 established in the presence of ^^C02, then ^'^COa was added 

 at zero time and illumination continued for a specified 

 interval until the cells were rapidly killed by immersion 

 in boiling alcohol. Subsequently the cell contents were 

 analysed. Calvin and colleagues combined the tracer method 

 with analysis by paper chromatography and radioautography 

 and in this way succeeded in identifying many compounds 

 into which tracer was incorporated in green cells in the light. 

 During periods of illumination of several minutes' dura- 

 tion many carbon compounds showed activity including 

 fats and proteins. When the period of illumination was 

 shortened to 30 seconds fewer compounds showed activity 

 and these included alanine, aspartic acid, malic acid, pyruvic 

 acid, and phosphoglyceric acid (PGA). In order to distin- 

 guish which of these was most intimately concerned in 

 photosynthesis the period of illumination was decreased still 

 further, it being argued that the shortest periods should 

 result in tracer appearing in only one compound, the first 

 product of photosynthesis. With periods of less than 2 

 seconds most of the tracer appears in PGA. Further, as the 

 period of illumination is shortened the percentage of tracer 

 carbon incorporated in PGA compared with the total tracer 

 incorporated rises, extrapolating to some 75% at zero time. 

 This probably indicates that PGA is the first intermediate 

 formed in detectable amounts. Of the remaining 25% malic 

 acid constitutes 3%, free glyceric acid 2%, phospho-enol- 

 pyruvic acid 3% and the rest consisting of small amounts 

 of glucose I -phosphate, sedoheptulose phosphate, fructose 

 monophosphate, and dihydroxyacetonephosphate. Some of 

 these compounds such as phospho-pyruvic acid show with 

 brief periods of illumination a very low percentage of tracer, 

 the percentage rising as the period of illumination is length- 

 ened but again decreasing as the period of illumination is 



