The following points highlight the four evidences for the Calvin cycle. The evidences are: 1. Evidence from Studies of Variation of Labeling of Intermediates with Time by Employing Radioactive 14CO2 2. Evidence from the Labeling Pattern of the Carbon Chain in the Intermediates 3. Evidence from Changes in the Steady-State Levels of Intermediates and 4. Evidence from Isolation of the Enzymes.
1. Evidence from Studies of Variation of Labeling of Intermediates with Time by Employing Radioactive 14CO2:
Melvin Calvin performed a series of such experiments using suspension of green algae, Chlorella and Senedesmus. A suspension of these algae was allowed to photosynthesize under conditions of constant temperature and illumination. Radioactive 14CO2 was then administered for a given period of time and the algal cells were subsequently killed by forcing them into boiling methanol.
The latter also served to extract the radioactive labelled compounds. These compounds in the alcoholic extract were separated from each other by paper chromatography and identified. Then, radioactivity in these compounds was determined. The incorporation of radioactive Carbon was found to be linear with time of exposure.
After an exposure of 30 sec. to 14CO2, radioactive carbon was found in 3-PGA, triose phosphates and hexose phosphates. But, when the time of exposure was reduced to 5 sec., the radioactivity was found only in 3-PGA. These experiments suggested 3-PGA to be the first stable carboxylation product of photosynthesis and that other triose phosphates and hexose phosphates were formed later.
2. Evidence from the Labeling Pattern of the Carbon Chain in the Intermediates:
After isolation and separation of labelled compounds, their molecules were degraded so that radioactivity could be determined in each carbon atom separately. In 3-PGA, radioactivity was found to be localised in carboxylic carbon. And because 3-PGA was primary carboxylation product, it was inferred that a 2C compound condensed with CO2 (to form 3C compound 3-PGA). But, intensive search for the existence of such a 2C compound by Calvin and his group failed.
Re-examination of early products of photosynthetic 14CO2 fixation by A.A. Benson in Calvin’s laboratory, showed that the chromatograms contained not only triose phosphates and hexose phosphates but also labelled pentose (5C) and heptose (7C) phosphates including 5C-ribulose 1, 5 bisphosphate (RuBP). This opened up the new possibility which was later proved, that the CO2 combined with a pentose bisphosphate (RuBP) to form a 6C compound which then cleaved into 2 molecules of 3-PGA.
In F-6-P (hexose phosphate), carbon no. 3 and 4 were found to be heavily labelled suggesting thereby that hexose phosphate were formed by condensation of 2 trioses.
By a thorough study of the radioactive carbon labelling pattern of pentose and heptose phosphate, it was concluded that 3-PGA was the starting material for their synthesis. Therefore, 3-PGA was precursor not only of hexose but also of RuBP.
The reaction sequences were then formulated for the synthesis of heptose and pentose phosphates which were at first hypothetical but later proved to be correct when the enzymes catalysing various postulated steps were isolated from plant cells.
3. Evidence from Changes in the Steady-State Levels of Intermediates:
As mentioned earlier RuBP is initial acceptor of CO2 and 3-PGA and RuBP are related in a cyclic manner in the Calvin Cycle. That this is so was confirmed by an experiment by Calvin by using green alga Chlorella. The algal cells were allowed to photosynthesize in presence of 14CO2 until steady conditions were obtained.
A steady state condition probably exists when the cells are illuminated i.e., 3PGA and RuBP are continuously being formed and broken down. Under such a condition any change in the conc. of intermediates can be measured by determining their radioactivity. A change from light to dark produced marked changes in the levels of 3- PGA and RuBP. Fig. 11.21. shows the effects of turning off and turning on of light on the levels of 3-PGA and RuBP.
When the light was turned off, there was a sharp increase in the level of 3-PGA and a fall in the level of RuBP. It is because in absence of light the supply of NADPH and ATP is cut off and in their absence 3-PGA cannot be converted into 3-phosphoglyceraldehyde either to form hexose phosphates or to regenerate RuBP. Although RuBP remained at low level throughout the dark period but 3-PGA showed a steadily fall from its high value probably due to its conversion to other metabolites.
When the light was again turned on, there was a sharp decrease in the level of 3-PGA with a consequent sharp increase in the level of RuBP until steady-state levels were again obtained. It is because in presence of light. NADPH and ATP were again produced and with their help accumulated 3-PGA was converted into triose phosphates, hexose phosphates and ultimately also into RuBP.
4. Evidence from Isolation of the Enzymes:
At first the reactions of the Calvin cycle were hypothetical, but later they were all found to be correct due to isolation of all the enzymes of Calvin cycle from chloroplasts of plant cells.