How did the use of ¹⁴C and autoradiography help Calvin uncover the pathway of carbon fixation?

Sourav PanNovember 9, 2024

Answered

Melvin Calvin’s experiments, particularly the “lollipop experiment,” utilized radioactive carbon-14 (¹⁴C) and autoradiography to elucidate the pathway of carbon fixation in photosynthesis. Here’s how these techniques contributed to understanding the carboxylation of ribulose bisphosphate (RuBP) and the overall Calvin cycle:

Use of ¹⁴C in Calvin’s Experiment

1. Introduction of Radioactive Carbon:
   • Calvin introduced ¹⁴C into a closed system containing green algae (Chlorella), which were placed in a specially designed apparatus known as the “lollipop.” This setup allowed for controlled exposure to light while ensuring that the radioactive carbon dioxide could be effectively incorporated into the algal cells during photosynthesis.
2. Carbon Fixation Tracking:
   • As light was shone on the apparatus, the algae underwent photosynthesis, incorporating the radioactive carbon into organic molecules. By using ¹⁴C, Calvin was able to trace the path of carbon as it was fixed from CO₂ into various intermediates during the process.

Autoradiography and Chromatography

3. Sample Collection and Quenching:
   • After specific time intervals, samples of the algae were rapidly killed using heated alcohol, which denatured proteins and stopped metabolic processes. This step ensured that any carbon compounds formed during photosynthesis were preserved for analysis.
4. Separation of Compounds:
   • The samples were then analyzed using two-dimensional chromatography, which separated different carbon compounds produced during photosynthesis. This technique allowed Calvin to isolate various intermediates formed at different time points.
5. Identification of Radioactive Compounds:
   • Autoradiography was employed to detect the presence of ¹⁴C in the separated compounds on the chromatogram. By exposing the chromatogram to X-ray film, Calvin could visualize where radioactive carbon had been incorporated into different molecules.

Mapping the Carbon Pathway

6. Determining Sequence of Reactions:
   • By comparing samples taken at various time intervals, Calvin was able to map out the sequence in which carbon compounds were generated during photosynthesis. This comparison revealed how CO₂ was converted into organic molecules through a series of intermediates, ultimately leading to carbohydrate production.
7. Elucidation of the Calvin Cycle:
   • The results showed that RuBP is carboxylated by CO₂ to form 3-phosphoglycerate (3-PGA), which is then further reduced to glyceraldehyde 3-phosphate (G3P). This process laid the groundwork for understanding the entire Calvin cycle, including how energy from ATP and reducing power from NADPH are utilized.