[13, XII. 1952J 



M. Calvin and P. Massini: The Path of Carbon in Photosynthesis 



453 



I ^ SCCNCKSMUS 



Fig. 9. — Radiogram of a paper chromatograra from lu min. 0^*0^ 

 fixation in light by ScCTi^iitfSmui. I % suspension, 1% Ct)j in air; light 

 intensity 7 x 10* ergs./cm^-s. D ll.A.P. ;dihydroxyacetone phosphate; 

 P.E.B. :phosphoeno]pyruvir acid; P.M.P.:pentose monophosphates; 

 P.Go.A.:phosphoglycolicacid: P.G.A.:phosphoglycericacid; H.M.P.: 

 hexose monophosphates; U.P.rpentose and hexose diphosphates. 



of the pair of stopcocks at H, and samples of 20 cm' of 

 the suspension withdrawn at intervals of five or ten 

 minutes. These samples were dropped into 80 cm' of 

 alcohol of room temperature, to make an e.xtraction 

 in 80% alcohol. After 30 min of photosynthesis, the 



lights were turned off and the suspension allowed to 

 remain in the dark for a period of 5 min, during which 

 time again several samples were withdrawn, and treated 

 in the same manner. In one experiment another light 

 jseriod followed the dark period. 



The samples were shaken for 1 h and centrifuged. 

 The residue was re-extracted in 50 cm' of 20% alcohol 

 at room temperature, centrifuged, and re-extracted 

 again with 20 cm' of water. The extracts were concen- 

 trated together to 0-5 cm'. 



An aliquot of the concentrate equivalent to 30 /il of 

 packed cells was evaporated on a corner of a filter paper 

 (Whatman #1), and the chromatogram run with water- 

 saturated phenol in one direction and n-butanol- 

 propionic acid-water in the other. The chromatograms 

 were exposed to X-ray film for about two weeks'. The 

 labeled compounds appeared on it as black spots. 

 Figure 9 shows the radiogram for ten minute photo- 

 synthesis of Scenedesmus. The amount of radioactivity 

 contained in the different compounds was determined 

 by counting the corresponding spots on the paper di- 

 rectly with a large-area Geiger-MCller tube with 

 thin mica window. The compounds were identified by 

 a combination of the following criteria: (a) Their posi- 

 tion on the paper; (b) the spot was cut out, eluted from 

 the paper with water and run again in suitable solvents, 

 together with such an amount of the suspected com- 



LIGHT 



TIME (min.) OF EXPOSURE TO 



Fig. 10. — C'*0, fixation by Scenedesmus. 1 % suspension, 1 % COg in 

 air, light intensity 7 x 10* ergs./cm*-s. 



JO 

 DARK 2 

 TIME (Kiln.) OF EXPOSURE TO (?*0j 



Fig. 11.— Behavior of radioactivity in specific compounds in the ex- 

 tract from the experiment of Figure 10. 



pound that it could be detected by a specific spraying 

 reagent. The black spot on the film had to coincide 

 accurately with the color reaction ; (c) the eluted spot 

 was chemically transformed {e.g. treating the sugar 

 phosphates with phosphatase) and the resulting com- 

 pound cochromatographed with carrier detectable by 

 spray. 



Figure 10 shows the total and the extracted amounts 

 of radiiKarbon fixed by 1 cm' cells during 30 min of 



' M. Calvin (. Chem. Education ?6, 639 (1949). 



87 



