164 - The Cell 



many teams of workers, in many laboratories. 

 But it is fair to say Melvin Calvin and col- 

 laborators at the Radiation Laboratory of the 

 University of California have been outstand- 

 ing leaders in this field — as was duly recog- 

 nized by the Nobel Prize award in 1961. 



Techniques. Two important technical devel- 

 opments contributed greatly to the prosecu- 

 tion of the carbon-tracing work. The dis- 

 covery and preparation of radioactive car- 

 bon (C 14 ), by Samuel Ruben and Martin 

 Kamen in 1940, provided an excellent tracer, 

 because radiocarbon is very stable (its half- 

 life is more than 5000 years) and because the 

 radioactivity provides a means of localizing 

 compounds in which C 14 is a constituent. But 

 almost equally important was the develop- 

 ment of paper chromatography, initiated by 

 two English workers, A. J. P. Martin and 

 R. L. M. Synge. 



Paper chromatography provides a method 

 for achieving the separation and identifica- 

 tion of unknown compounds, when these are 

 dissolved in small amounts in an aqueous 

 extract of cellular material. The separation 

 results from small differentials in the solubil- 

 ities of the compounds, as is indicated in Fig. 

 9-5. A drop of the aqueous solution of un- 

 known compounds (X, Y, and Z) is placed on 

 the filter paper, near the bottom, where the 

 paper is in contact with a suitable organic 

 solvent, saturated with water. As the organic 

 solvent creeps by capillarity upward in the 

 paper, it first will pick up compound X — if 

 this displays the greatest degree of solubility 

 in the particular organic solvent — and com- 

 pound X will be carried the greatest distances 

 from the locus of the original spot (Fig. 9-5). 

 Likewise Y and Z, if this is the sequence of 

 decreasing solubility, will not be picked up 

 as quickly or carried as far by the upward- 

 flowing solvent (Fig. 9-5). A further two- 

 dimensional separation of the components 

 can also be achieved by a second-stage treat- 

 ment in which the position of the paper is 

 changed, as is explained in the caption of 

 Figure 9-5. 



Identification of the separated compo- 



0IRECTI0N OF 



SOLVENT 



MOVEMENT 



ORIGINAL AQUEOUS 

 SPOT CONTAINING 

 MIXTURE OF 

 UNKNOWN SUBSTANCES(X,V,Z) 



DISH WITH SOLVENT 



Fig. 9-5. One-dimensional (ascending) paper chro- 

 matography. The creeping solvent carries the sub- 

 stances at different rates, the final distances (note 

 arrows) being inversely related to water solubility. 

 Thus substance X (e.g., triose phosphate) would be 

 carried further than Y (e.g., phosphoglyceric acid), 

 which in turn would be carried further than Z (e.g., 

 hexose diphosphate). Further separation of the sub- 

 stances can be effected by a second stage of treat- 

 ment (two-dimensional chromatography) in which the 

 position of the paper strip is changed. In the present 

 case, for example, the left side of the paper might be- 

 come the bottom, which is in contact with the solvent. 

 Moreover, a different organic solvent may be used in 

 the second stage. 



nents requires further operations. Radioac- 

 tive materials may be localized by plating the 

 paper upon photographic film; but more spe- 

 cific detection requires that the paper be 

 sprayed with reagents, which bring about 

 some specific color reaction, or it may in- 

 volve other analytical procedures. 



Pathway of Carbon in Organic Synthesis: 

 The Calvin Cycle. The pioneer experiments 

 of the Calvin group regularly utilized a uni- 

 cellular green alga Chlorella (Fig. 9-9) al- 

 though confirmatory results have been de- 

 rived from higher plants. All essential condi- 

 tions in the experiments — intensity and 

 duration of illumination, density of the cell 

 population, temperature, concentration, and 

 time of the exposure to radioactive carbon 

 dioxide (C 14 2 ), and so forth — were kept 

 under strict control. Immediately after the 

 experimental treatment, which sometimes 

 lasted only a few seconds, the cells were 

 quickly killed in alcohol, extracted, and 

 analyzed by paper chromatography for their 

 content of newly synthesized material. 



It was surprising to find that even when 



