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The Calvin Cycle Was Determined by Isotope Labelling Methods

The steps in the Calvin cycle involve the conversion of one type of molecule to another, eventually regenerating the starting material, RuBP. In the 1940s and 1950s, Calvin and his colleagues used 14C, a radioisotope of carbon, as a way to label and trace molecules produced during the cycle. They injected 14C-labelled CO2 into cultures of the green algae Chlorella pyrenoidosa grown in an apparatus called a “lollipop” (because of its shape). The Chlorella cells were given different lengths of time to incorporate the 14C-labelled carbon, ranging from fractions of a second to many minutes. After this incubation period, the cells were abruptly placed into a solution of alcohol to inhibit enzymatic reactions and thereby stop the cycle.

The researchers separated the newly made radiolabelled molecules by a variety of methods. The most commonly used method was two-dimensional paper chromatography. In this approach, a sample containing radiolabelled molecules was spotted onto a corner of the paper at a location called the origin. The edge of the paper was placed in a solvent, such as phenol-water, and the solvent would ascend to the top of the paper. As the solvent rose through the paper, so did the radiolabelled molecules. The rate at which they rose depended on their structures, which determine how soluble they are in the solvent and how strongly they interact with the fibres in the paper. This step separated the mixture of molecules spotted onto the paper at the origin. The paper was then dried and turned 90 degrees, and then the edge was placed in a different solvent, such as butanolpropionic acid-water. Again, the solvent would rise through the paper, thereby separating molecules in a second dimension. Note that a specific molecule could be very soluble in one of the solvents and much less so in the second solvent. After this second separation step, the paper was dried and exposed to X-ray film, a procedure called autoradiography. Radioactive emission from the 14C-labelled molecules caused dark spots to appear on the film.

The pattern of spots changed, depending on the length of time that the cells were incubated with 14C-labelled CO2. If the incubation period was short, only molecules that were made in the first steps of the Calvin cycle were seen, while longer incubations revealed molecules synthesized in later steps. For example, after short incubations, 3-phosphoglycerate (3PG) and 1,3-bisphosphoglycerate (1,3-BPG) were observed, while longer incubations showed glyceraldehyde-3-phosphate (G3P) and ribulose bisphosphate (RuBP).

A challenge for Calvin and his colleagues was to identify the chemical nature of each spot. This was achieved by a variety of chemical methods. For example, a spot could be cut out of the paper, the molecule within the paper could be washed out, or eluted, and then the eluted molecule could be subjected to the same procedure that included a radiolabelled molecule whose structure was already known. If the unknown molecule and known molecule migrated to the same spot in the paper, this indicated that they were the same molecule. During the late 1940s and 1950s, Calvin and his coworkers identified all the 14C-labelled spots and the order in which they appeared. In this way, they were able to determine the series of reactions of what we now know as the Calvin cycle. For this work, Calvin was awarded the Nobel Prize in 1961.
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