APPLICATION OF PAPER CHROMATOGRAPHY 1661 



synthesize for an hour in normal carbon dioxide in the presence of radio- 

 active phosphate. Active spots were found in the same region near the 

 origin of the radiogram (fig. 36.15). A similar pattern could be produced 

 also by using P (32) -labelled phosphorylated intermediates from the fer- 

 mentation of glucose by yeast. By separating the several phosphate esters 

 from the fermentation product (by selective elution from an anion exchange 

 resin) and chromatographing them in one dimension, it was ascertained 

 that the phosphoglyceric acid moves ahead of fructose-6-phosphate, and 

 the latter moves ahead of fructose-l,6-diphosphate. The three above-men- 

 tioned spots in C(14) radiograms from photosynthesizing cells were there- 

 fore identified, from the bottom upward, as ''hexose diphosphate," "hexose 

 monophosphate" and "phosphoglyceric acid." 



Since the third spot was the earliest to appear, and therefore com- 

 manded the greatest interest, experiments were made to support its identi- 

 fication as phosphoglyceric acid. P(32)-labelled material from algae 

 which had been exposed for 1 hr. to ordinaiy CO2 in active phosphate was 

 coprecipitated with synthetic barium phosphoglycerate, and the precipi- 

 tate, after conversion to free acid, was cochromatographed with the ex- 

 tract obtained from algae after 90 sec. photosynthesis in C*02. The perim- 

 eters of the P* and C* activities in the region of the "third spot" were 

 found to coincide, thus indicating that the C*-labelled compound must be 

 identical with the P*-labelled compound (whose barium salt was proved 

 before to be coprecipitable with barium phosphoglycerate). 



From products of 5 sec. exposure of Scenedesmus cells to C*02 (which, 

 we recall, gave one strong radioactive spot only), phosphoglyceric acid was 

 isolated and identified by the following procedure: 1-g. batches of cells, 

 exposed for 5 sec. to C*02 in intense light, were added to 18 g. normal algae 

 (to provide bulk for isolation). The algae were killed by acid (4CH3- 

 COOH, glacial + 1 HCl, cone), all activity going into aqueous extract. 

 (This method was again used, instead of killing with 80% alcohol, to re- 

 move as much protein, cellulose and lipids as possible at the very beginning 

 of the process.) 



Fractionation of the extract by precipitation at pH 7 and leaching out 

 at pH 10, repeated precipitation with BaCl2 from 50% ethanol, and dissolu- 

 tion in 0.05 A'^ HCl, led to a crystalline precipitate whose P content and 

 specific rotation were close to those of barium-3-phosphogly cerate. Oxida- 

 tion by periodate led to products (formaldehyde, formic acid, carbon 

 dioxide) found also with glyceric acid. The somewhat lower rotation of 

 the photosynthetic product was taken as indicating the presence of a 

 small amount of 2-phosphoglyceric acid of higher specific activity than the 

 main amount of 3-phosphoglyceric acid. On some of the radiograms, 

 the PGA spot was in fact double; the "right half" could be changed 



