Further Links Contact Us Search Help

Syllabus | Exams | Exam techniques | Resources | Teachers | Feedback

Biology

Home > Biology > Options > Biochemistry > Biochemistry: 6. Using isotopes to prove carbon dioxide is used in the light independent reaction

Option 9.9 Biochemistry: 6. Using isotopes to prove carbon dioxide is used in the light independent reaction

Syllabus reference (October 2002 version)
6. The discovery of C14 in the mid 20th century allowed a detailed study of the role of carbon dioxide in photosynthesis	Students learn to: identify that Kamen and Ruben discovered C14 and demonstrated that radioactive carbon dioxide could be used to investigate the chemical transformations of carbon dioxide during photosynthesis in 1940 describe the experiments, using paper chromatography, that Calvin carried out to deduce the products of photosynthesis   outline the main steps of the Calvin cycle as: the production of phosphoglycerate from the combining of carbon dioxide with an acceptor molecule the reduction of phosphoglycerate into glyceraldehyde phosphate in two reactions that use ATP and NADPH produced in the light reactions the regeneration of the initial carbon dioxide   explain why the Calvin cycle is now called the light independent stage of photosynthesis	Students: gather and process information to use a biochemical pathways chart to trace the steps in the Calvin cycle

Extract from Biology Stage 6 Syllabus (Amended October 2002) © Board of Studies, NSW.
[Edit:18 June 09]

Prior learning: Stage 4, Models, Theories and Laws 4.8.1(c) Structures and Systems 4.8.2 (c), 4.8.4(d) Interactions 4.10 (c); Stage 5, Models, Theories and Laws 5.6.5 (a) and (b), 5.7.1.

Recall statements in Preliminary course: Preliminary module 8.2 (subsection 2) module 8.3 (subsection 4).

Background: The discovery of C₁₄ in the mid 20th century allowed a detailed study of the role of carbon dioxide in photosynthesis.

identify that Kamen and Ruben discovered C14 and demonstrated that radioactive carbon dioxide could be used to investigate the chemical transformations of carbon dioxide during photosynthesis in 1940

• Sam Ruben at the University of California, Berkeley, pioneered the concept of using radioactive carbon to trace the early events of CO₂ fixation. As early as 1939, Ruben had been feeding ¹¹CO₂ to the single cell algae Chlorella. The short lifetime of the isotope (21 minutes) prevented a clear result. In collaboration, Martin Kamen and Sam Ruben succeeded in discovering and preparing sufficient ¹⁴C to determine its half life as 5,700 years. Ruben was killed in a laboratory accident before large-scale methods for production of ¹⁴C were practical and Kamen was dismissed from the Berkeley Laboratories as a security risk. They had discovered and pioneered the use of ¹⁴C in biochemical reactions but the work was to be carried on by Melvin Calvin and his co-workers.

describe the experiments, using paper chromatography, that Calvin carried out to deduce the products of photosynthesis

• The experiments carried out by Calvin and his co-workers involved an illuminated “lollipop” in which Chlorella (unicellular photosynthetic algae) was placed. The “lollipop” was a flattened glass vessel, the shape of a large lollipop on a stick, wide but thin. The algae were given a pulse of radioactive carbon dioxide (¹⁴CO₂), injected into a stream of air containing non-radioactive CO₂ for a suitable period.
• Samples of the Chlorella were then released at intervals (3, 5, 10 seconds and then 15 second periods) into boiling alcohol to stop the progress of ¹⁴C at different moments in the metabolic pathway for the synthesis of glucose. Compounds that the radioactive carbon had reached at a particular moment were determined by two dimensional paper chromatography and autoradiography.
• Each sample was separated using paper chromatography (see 9.9 Biochemistry: 3. The discovery of the chloroplast as the site of photosynthesis) and the components identified. Placing the chromatogram over a sheet of X-ray film identified those that contained the ¹⁴C. The radioactivity exposed the film and so the movement of the ¹⁴C could be traced (linked to various compounds over time).

outline the main steps of the Calvin cycle as:

• the production of phosphoglycerate from the combining of carbon dioxide with an acceptor molecule
• the reduction of phosphoglycerate into glyceraldehyde phosphate in two reactions that use ATP and NADPH produced in the light reactions
• the regeneration of the initial carbon dioxide acceptor

• The earliest labeled product from Calvin’s experiments was phosphoglycerate (3-phosphoglycerate or 3PG) formed by the addition of CO₂ to a 5-carbon acceptor. The resulting 6-carbon compound is broken into two molecules.

CO₂ + 5-carbon acceptor → [6-carbon intermediate] → two phosphoglycerate.

• In the next step a phosphate group from ATP is added to each molecule of phosphoglycerate (3PG) to form an intermediate called 1,3-diphosphoglycerate.
• In the next step a pair of electrons donated by NADPH reduces this intermediate to form glyceraldehyde phosphate.
• Glyceraldehyde phosphate is a 3-carbon sugar, not the 6-carbon sugar glucose generally identified as the end product of photosynthesis. Glyceraldehyde phosphate leaves the Calvin cycle and is converted to glucose and stored as starch in the stroma of the chloroplast or used in other reactions in the cytosol to make sucrose for transporting to other parts of the plant.
• The regeneration of the initial 5-carbon acceptor in the Calvin cycle avoids wasteful reactions that use large amounts of ATP and NADPH and allows continuous CO₂ fixation. In order to regenerate the 5-carbon acceptor the cycle runs three times.

3C5 + 3C1    > 3C6 (unstable)    > 6C3.

One of these C3 molecules leaves the cycle and the other five C3 molecules are regenerated to form 3 molecules of the C5 acceptor.

Counting C atoms:	3C x 5 + 3C x 1 = 18C 3C x 1 leaves the cycle = 3C 3C x 5 regenerated = 15C

• The net equation is

3CO₂ + 9ATP + 6NADPH + water → glyceraldehyde phosphate + 8P_i + 9ADP + 6NADP⁺.

The ATP and NADPH come from the light dependent reactions, the inorganic phosphate (P_i), the adenosine diphosphate (ADP) and nicotinimide adenosine diphosphate are recycled to the light reactions

gather and process information to use a biochemical pathways chart to trace the steps in the Calvin cycle

• Gather information from a range of resources including the ones listed below.

Biochemistry texts,
Molecular Biology of the Cell; Alberts, B. et al. Garland Publishing: or Biology; (3^rd edition) Campbell, N.A. The Benjamin /Cummings Publishing Company, or scientific journals to find three copies of the biochemical pathways of the Calvin Cycle.
Use the term Calvin cycle in a search engine such as Google (www.google.com) to find biological pathways of the Calvin cycle or use the pages below.

Calvin cycle J Kimball, USA.

The Calvin Cycle Process , Michigan State University, Michigan, USA.

• Process the information by assessing the reliability of the information from various sources. Choose the version of the cycle (they will all be very different!) that you find best illustrates the reactions of the cycle using the information given above.

explain why the Calvin cycle is now called the light independent stage of photosynthesis

• Benson (and Edward McMillan) demonstrated that CO₂ fixation was independent of light by illuminating on a sample of algae in the absence of CO₂, immediately transferring the algae to a black flask containing ¹⁴CO₂ and analysing the products for radioactivity. Sucrose (formed from glyceraldehyde phosphate) contained radioactive ¹⁴C was isolated at a rate approaching it’s formation in light. This experiment proved that the energy absorbed by chlorophyll was used for the production of phosphorylating (ATP) and reducing agents (NADPH) capable of driving the conversion of CO₂to sugar in the dark.

• Although the fixation of carbon does not require light, it does not take place in the dark! The enzymes of the Calvin cycle respond indirectly to light activation. When light energy is available to generate ATP and NADPH, the Calvin cycle proceeds. In the dark, when ATP and NADPH cannot be produced by the light dependent reaction, fixation of CO₂ ceases. Therefore, the Calvin cycle is now called the light independent stage of photosynthesis.

• The enzyme that catalyses the capture of CO₂ from the atmosphere by the 5-carbon acceptor (ribulose bis-phosphate, abbreviated to RuBP) is called RuBP carboxylase or rubisco. It is an extremely large enzyme that works very slowly, 3 molecules of substrate per second, compared to the normal 1000 molecules of substrate for most enzymes. Many copies are therefore needed and rubisco constitutes more than 50% of the total protein in chloroplasts and is thought to be the most abundant protein on earth.

• Melvin Calvin, Andrew Benson and James Bassham determined the series of biochemical reactions in the Calvin cycle in the late 1940s and 1950s. It was completed in 1958 and earned Melvin Calvin the Nobel Prize for Chemistry in 1961. Calvin recognised the fact that the CO₂ was added to a 5-carbon acceptor to form a 6-carbon intermediate that instantaneously split in half. Benson and Bassham collaborated to identify the substances.