• Enzyme controlled (Q₁₀ » 2)
  
• Located in the stroma of the chloroplast.
  
• Occurs simultaneously with the light dependent reaction but it can continue in the dark provided the necessary raw materials are available (CO₂, NADPH + H⁺ and ATP)
  

To find out the sequence of the reactions and the point at which X is added in, two approaches can be used:

1. Label and trace the products formed through time.
   
2. Cut the supply of X and observe what happens to the intermediates in the pathway (e.g. Photosynthesis cut the CO₂ supply or switch off the light, thus, cutting the supply of ATP and NADPH+H⁺)

Calvin and Benson 1946 to 1953

• Used ¹⁴C radioisotope for labelling (available after WW II)

• Unicellular algae: Chlorella and Scenedesmus.
  Simple plants which respond quickly to changes in the environment. So little time lag.

"Lollipop" vessel. A flat-sided, round flask containing the culture of algae
This shape:

Add NaH¹⁴CO₃ solution.

At timed intervals the algae are sampled and killed by dropping in hot methanol.

Two-way (2-dimensional) chromatography used to separate the compounds.

Identify radioactively labelled compounds by autoradiography.

The compound that became labelled first was Phosphoglyceric Acid (or Phosphoglycerate) PGA.

PGA is a 3 carbon sugar.

Therefore: Compound B + CO₂ PGA

Feed the algae NaH¹⁴CO₃ for over 15 min so all the compounds involved in photosynthesis are labelled.

Then reduce the level of NaH¹⁴CO₃ rapidly.

PGA levels should fall rapidly

The compound that fixes CO₂ should rise sharply.

This compound was found to be Ribulose Bisphosphate (RBP)

RBP is a 5 carbon sugar

Therefore

The enzyme controlling this important step is Ribulose Bisphosphate Carboxylase (Rubisco)

The light dependent pathways produce ATP and NADPH+H⁺ to be used in the light independent processes.

Illuminate the algae for 15 min then cut the light.

PGA levels rise

Other compounds, after PGA in the pathway, fall

The compound requiring the NADPH+H+ and ATP was found to be Glycerate 3-phosphate (GP)

GP is converted to various other three carbon sugars or Triose Phosphates (TP) some are used to produce hexoses such as fructose phosphate and glucose phosphate.

When the light is cut interestingly RBP levels also fall.

This is for two reasons.

• RBP requires ATP in its synthesis
• RBP is formed from some of the GP. In other words the reaction pathway is a CYCLE.