What was Calvin’s experiment, and how did it elucidate the carboxylation of RuBP?

Sourav PanNovember 9, 2024

Answered

Calvin’s experiment, commonly referred to as the “lollipop experiment,” was instrumental in elucidating the carboxylation of ribulose bisphosphate (RuBP) during the Calvin cycle of photosynthesis. This series of experiments conducted by Melvin Calvin and his colleagues in the 1950s employed radioactive carbon-14 to trace the path of carbon as it was incorporated into organic molecules. Here’s a detailed overview of the experiment and its significance:

Overview of Calvin’s Experiment

1. Apparatus Design:
   • The experiment utilized a unique apparatus resembling an upside-down lollipop, which allowed for the introduction of carbon dioxide (CO₂) into a chamber containing green algae, specifically Chlorella. The design facilitated controlled exposure to light while maintaining a closed system for gas exchange.
2. Use of Radioactive Carbon-14:
   • Radioactive carbon-14 was introduced into the apparatus as the sole source of carbon. This allowed Calvin to track how CO₂ was incorporated into organic compounds during photosynthesis. Light was shone on the algae to stimulate photosynthesis, promoting the incorporation of carbon-14 into various intermediates.
3. Sampling and Quenching:
   • After specific time intervals, samples were taken from the algae and immediately treated with heated alcohol to halt metabolic processes. This step ensured that any carbon compounds formed during photosynthesis would be preserved for analysis.
4. Analysis via Chromatography:
   • The samples were analyzed using two-dimensional chromatography, which separated different carbon compounds produced during photosynthesis. Autoradiography was then employed to detect the radioactive carbon-14 in these compounds, allowing Calvin to identify which molecules were formed at different time points.
5. Mapping Carbon Flow:
   • By comparing samples taken at various intervals, Calvin was able to map out the sequence in which carbon compounds were generated during photosynthesis. This led to the identification of key intermediates in the Calvin cycle, particularly those involved in the carboxylation of RuBP.

Elucidation of Carboxylation of RuBP

1. Carboxylation Reaction:
   • The primary finding from Calvin’s experiments was that RuBP serves as the initial acceptor of CO₂ in the Calvin cycle. The enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes this reaction, resulting in a six-carbon intermediate that quickly splits into two molecules of 3-phosphoglycerate (3-PGA).
2. Identification of Intermediates:
   • Through his experiments, Calvin identified several key intermediates formed during the conversion of CO₂ to carbohydrates, including 3-PGA and glyceraldehyde 3-phosphate (G3P). These findings provided insight into how inorganic carbon is fixed and transformed into organic molecules.
3. Proposing the Calvin Cycle:
   • The results from these experiments laid the groundwork for what is now known as the Calvin cycle, detailing how CO₂ is fixed into organic compounds through a series of enzymatic reactions involving RuBP and its subsequent regeneration.