1GG2 CHEMICAL PATH OF CARBON DIOXIDE REDUCTION CHAP. 36 



into the "left half" by heating in 0.1 A^ HCl for 1 hr. It was suggested 

 that the right half is 2-phosphoglyceric acid and the left one, 3-phospho- 

 glyceric acid. The proportion of the 2-substituted acid was much higher in 

 strong than in weak light. When the temperature during the C*02 ex- 

 posure was lowered to 4° C, only the right half of the spot appeared. It 

 thus seems that the 2-phosphoglyceric acid is the first formed, but less 

 stable isomer. 



The crystalline product which thus had many properties of authentic 

 phosphoglyceric acid weighed 10 mg. and accounted for one-third of total 

 C* fixed in 5-sec. exposure. The last washings and supernatants from this 

 preparation accounted for another 30%; when chromatographed, they, 

 too, deposited their activities in the PGA spot. 



Easily hydrolyzable phosphate esters could be identified in the paper 

 . chromatograms by spraying with molybdate to form molybdenum blue. 

 In this way, glucose-1-phosphate, phosphopyruvic acid, and triose phos- 

 phate (apparently, both 3-phosphoglyceraldehyde, and phosphodihydroxy- 

 acetone) were identified in positions shown in figure 36. 13. Phosphopyruvic 

 acid is one of the earliest of these compounds to appear in photosynthesis, 

 as illustrated by figure 36.6a. According to Calvin et al. (1950), free 

 glyceric acid often appears, together with malic acid and phosphopymvic 

 acid, as the earliest companions of phosphoglyceric acid in the radiograms 

 from briefly photosynthesizing cells. In one experiment, after 5-sec. photo- 

 synthesis, 87% of the tracer was found in PGA, 10% in PPA and 3% in 

 malic acid. These results agree with Gaffron and Pager's observations 

 (Fig. 36.10), except that the latter listed PA rather than PPA as the main 

 companion of PGA in the first seconds of photosynthesis. In all probabil- 

 ity, both PGA and PPA may undergo hydrolysis in the extraction and sep- 

 aration processes; free acids, GA and PA, will then be found instead of the 

 originally present phosphate esters. 



The identification and order of appearance of amino acids was men- 

 tioned above. It is significant that some of them — those with C3 and C4 

 chains— appear very early, even before tagged sugars or their phosphate 



esters. 



With these paper-chromatographic observations, the previous results, 

 obtained with ordinary analytical methods and summarized in tables 

 36. Ill and 36.IV, were largely confirmed and greatly enlarged. The role 

 of phosphoglyceric acid as the first compound which becomes tagged in 

 photosynthesis was established much more firmly than before. The 

 early appearance of phosphopyruvic acid and of the phosphates of trioses 

 and hexoses was clarified. (In previous experiments, all these compounds 

 must have been hidden in the second anionically tagged, "non ammonia- 

 elu table," subfraction.) The early appearance of tagged amino acids was 



