448 



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



[ExperientiaVol.VIII/12] 



relatively short periods of time most of the fixed radio- 

 activity is found in the soluble components, the prob- 

 lem is one of analyzing for the distribution in the 

 soluble fraction. This has been done by an application 

 of the method of paper chromatography introduced 

 and developed for amino acid analysis by Consden, 

 Martin, and Synge'. It has since been applied to a 

 wide variety of compounds and no detailed description 

 of it will be given here. The unique extension to our 

 work lies in the ability to locate particularly those 

 compounds which contain the radioactive carbon atoms 

 on the paper by means of a radioautograph of the 

 resulting paper chromatogram obtained by allowing an 

 X-ray film to remain in contact with the paper for a 

 suitable period of time. Those areas of the paper which 

 are occupied by radioactive compounds will, of course, 

 expose the X-ray film. Such a map of the disposition 

 of the radioactive compounds contained in an extract 

 is shown in Figure 5. The chemical nature of the com- 

 pounds defined by the exposed areas can be inferred 

 from the position occupied by a compound with re- 

 spects to the origin of the chromatogram. More precise 

 determination of the chemical character is assisted by 

 chemistry performed on the material eluted from the 

 spot defined by the radiogram and rechromatography. 

 Final identification, however, is usually dependent on 

 the co-chromatography of the unknown, or questioned, 

 radioactive material eluted from the paper with an 

 authentic specimen of the suspected compound and 

 the demonstration of the complete identity of the car- 

 rier material as determined by some visible test on the 

 paper with the pattern of radioactivity in the co-chro- 

 matogram. The amount of radioactivity incorporated 

 in these compounds can be determined quite accurately 

 by using the X-ray film as a means of defining that 

 area of the paper containing the compound, thus per- 

 mitting the particular spot to be cut out from the larger 

 and eluted from the paper and mounted on a plate to 

 be counted. 



*f.^i=n8s»i:':r.--i' T.",i»W:' 



OMVOMTtMCTONK mOIMWTf 



wWBafSBSWBBSr'"^'^'-' 



\ PHO*n(0«4.Y0tllATt 



.^ 



.y 



mtUlOM mtOfHATE 



mtOM MOMHATI 

 MRMTOM mOVMTl ft MMMOtC mOWlun' f 



•LUOOM nWtMUTC ft ItlMMPTULOte MQtPHATI 



m 



•uftULOtc wmowtun • niiatB emioftnuTff 



Fig. 5, 



-Radiogram of a paper chromatogram from 10 s C**0, fixation 

 ID the light by Scenedesmus. 



* R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 

 28, 224 (1944). - A. A. Benson, J. A. Bassham, M. Calvin, T. C. 

 GooDALE, V. A. Haas, and W. Stepka, J. Am. Chem. Soc. 72, 1710 

 (1950). 



Fig. 6, 



— Behavior of radioactivity in specific compounds in extracts 

 Scenedestnus , exposed to radioactive carbon dioxide at 2*'C. 



A much simpler means would be to count the spot 

 right on the paper with a Geiger counter. The fraction 

 of the total amount of radioactivity in the spot which 

 is thus registered by the Geiger counter is fairly con- 

 stant for all compounds for any given chromato- 

 graphic system. Thus, for most purposes it is sufficient 

 simply to expose the paper to X-ray film in order to 

 determine just where the radioactive spots are, and 

 then having so defined them, to count them right on 

 the paper for quantitative comparison, by the Geiger 

 counter. It is clear from Figure 5 that the variety of 

 products synthesized at room temperature by Scene- 

 desmus (as well as by all other plants tried) is great, 

 even in a very short time such as ten seconds. But even 

 so, it is clear that the predominant compound as the 

 time gets shorter is phosphoglyceric acid. 



This is even more strongly demonstrated when the 

 experiment is carried out at reduced temperature, for 

 instance 2°C, so as to slow down all of the reactions 

 and enable us to see more clearly the earliest products. 

 Figure 6 shows a plot of the concentration of radio- 

 activity per unit of algae for three of the major early 

 compounds, formed at 2°C. On such a plot as this, it 

 is clear that those substances which are formed directly 

 from carbon dioxide with no appreciable intermediates 



82 



