ri5. XII. 1952] 



M. Calvin and P. >[assini: The I\itli u( Ciiboii in Pliolusyntheais. 



447 



molecules of water and the requisite number of light 

 quanta into a single unit followed by the rearrangement 

 into hexose and molecular oxygen, no specific infor- 

 mation was available as to the compounds which might 

 act as intermediates. Assuming that such a chain of 

 intermediates exists, it is quite clear that by setting up 

 some photosynthetic organism, leaf or other suitable 

 material, in a steady state of photosynthesis in which 

 the various ingredients are being absorbed and pro- 

 ducts formed in some uniform manner and injecting the 

 labeled carbon dioxide into the entering carbon dioxide 

 stream, we should find the label appearing successively 

 in time in that chain of intermediates. This can be 

 observed by stopping the entire process after a suitable 

 lapse of time and examining the incorporated labeled 

 carbon to determine the nature of the compounds into 

 which it has been built. It is also clear that in addition to 

 the identity and sequence of the compounds into which 

 the carbon is incorporated, we may also determine the 

 order in which the various carbon atoms within each 

 compound acquire the label. With this type of infor- 

 mation at hand it should be possible to reconstruct the 

 sequence of events from the time of entry of the carbon 

 atom into the plant as carbon dioxide until it appears 

 in the various more or less finished products of the plant . 



Fig. 3.— .\lg.ic I'l.iiU. 



While photosynthetic experiments have been done 

 with a vide variety of plant materials, the major ki- 

 netic work has been carried out with suspensions of 

 unicellular green algae. The reason for this lies in the 



I'ig. 4. — "Lollipop". 



fact that these algae may be obtained in a reproducible 

 biological form relatively easily and in any amount. 

 They are grown in the laboratory in a continuous cul- 

 ture arrangement shown in Figure 3. The algae maybe 

 harvested from these flasks daily or every other day, 

 depending upon the type of material desired. Such 

 cultures have been maintained in a continuous fashion 

 over periods extending beyond several months. Most 

 of our experiments have been performed with the uni- 

 cellular green algae Chlorella or Scenedesmus. After 

 harvesting the algae are washed with distilled water 

 and resuspended in the medium in which the experi- 

 ment is to be done. This suspension is placed in a flat 

 vessel called a "lollipop", a photograph of which is 

 shown in Figure 4. A stream of air containing carbon 

 dioxide is passed through the algae while they are 

 being illuminated so as to achieve a steady state of 

 photosynthesis. 



In order to begin the experiment the air stream is 

 interrupted and the labeled bicarbonate is injected into 

 the algal suspension. After the preselected period of 

 time, the algae are killed by opening the large stopcock 

 at the bottom of the flask, allowing the algal suspension 

 to fall into alcohol in order to stop the reaction and 

 extract the photosynthesized material. Although a 

 variety of killing and extracting procedures have been 

 tested, most of the experiments were performed by 

 dropping the algae into alcohol so as to result in an 

 80% alcohol solution. The total amount of carbon 

 fixed is then determined by taking an aliquot of this 

 entire suspension, evaporating it to dryness on an alu- 

 minum disk and counting it on a Geiger counter'. The 

 fraction soluble is determined by either filtering or 

 centrifuging the suspension and then recounting the 

 clear supernate or filtrate. 



The distribution of the fixed radiocarbon among the 

 various compounds must now be determined. Since in 



' M. Calvin, C. HtlutLUERGER, J. C. Reid, Lt. M. Tolbert, and 

 P. E. Yankwich, Iwtopic Carbon (John Wiley & Sons, Inc., New 

 York, 1940). 



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