Required practical 8
Investigation into the effect of a named factor on the rate of dehydrogenase activity in extracts of chloroplasts.
Overview
- Dehydrogenase is an enzyme in the Light Dependent Reaction
- It catalyses the transfer of hydrogen from one molecule to another during the light-dependent reaction
- In this practical, DCPIP acts as an artificial electron acceptor (replacing NADP) to measure this activity. It’s a redox indicator, so it changes colour when it is reduced.
- When DCPIP is oxidised it is blue, when it has accepted electrons it is colourless (reduced)
- We can measure the rate of dehydrogenase activity, by measuring the rate at which the indicator changes from oxidised (blue) to colourless
High Level Method
- Prepare a chloroplast extract using cell fractionation. This involves breaking down the leaf tissue in a suitable solution, then centrifuging to separate the chloroplasts from other cell components.
What three conditions does the solution need, and why?
What three conditions does the solution need, and why?
- Same water potential as the chloroplasts: no osmosis, so the chloroplasts (not the cells) don’t burst or shrivel
- Ice-cold: reduces the activity of enzymes that would damage the chloroplasts
- Buffered: keeps the pH constant so enzymes and proteins aren’t denatured
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Add a fixed volume of chloroplast extract to a fixed volume of DCPIP in test tubes and mix gently.
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Expose each test tube to a different light intensity, for example by placing them at different distances from a lamp (e.g. 30 cm, 50 cm, 100 cm)
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Set up control tubes: one with DCPIP but no chloroplasts (in the light), and one with chloroplasts and DCPIP kept in the dark.
Why set up these two control tubes?
Why set up these two control tubes?
- No chloroplasts: shows that light alone doesn’t make DCPIP change colour, so chloroplasts are needed
- In the dark: shows that the chloroplasts only reduce DCPIP when there is light
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Measure the absorbance of each sample using a colorimeter at regular intervals (e.g. every 2 minutes for 10 minutes).
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Determine the rate of dehydrogenase activity from the decrease in absorbance of DCPIP over time, comparing rates between light intensities.
Interpreting Results
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DCPIP is blue when oxidised and has a high absorbance
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When DCPIP is reduced it becomes colourless, so absorbance decreases
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The faster DCPIP becomes colourless, the higher the rate of dehydrogenase activity
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As the reaction occurs in the light-dependent stage of photosynthesis, increasing light intensity increases the rate of electron transfer to DCPIP, so DCPIP decolourises faster
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Example graph with conditions below
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A = steepest decrease: fastest rate
- Highest light intensity (e.g. nearest to the lamp)
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B = moderate decrease: medium rate
- Moderate light intensity (e.g. not the nearest to the lamp but still receives enough light)
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C = shallow decrease: slowest rate
- Shade/low light intensity
See also: Chromatography of leaf pigments (required practical 7).
Dehydrogenase activity is examined as part of photosynthesis. See how it's tested and the tips from examiner reports on that page →
Exam Question Practice
A student isolated chloroplasts from pea leaves into a solution to form a chloroplast suspension. The solution had the same water potential as the chloroplasts.
Explain why it was important that these water potentials were the same.
(2 marks)
Hint
What would water do if the water potentials were different? What would happen to the chloroplasts?
Mark Scheme
- Osmosis does not occur (1 mark)
- Chloroplast/organelle does not burst/lyse/shrivel/shrink (1 mark)
Also accepted / rejected
Tips
- Describe the effect on the chloroplasts (they would burst or shrink), not on ‘the cell’
- Don’t use ‘plasmolysed’ or ‘turgid’ for chloroplasts
A student used a chloroplast suspension from pea leaves and DCPIP solution to investigate the light-dependent reaction of photosynthesis. DCPIP solution is blue when oxidised and colourless when reduced.
The student set up three test tubes as follows:
- Tube A – 2 cm³ of chloroplast suspension and 8 cm³ of DCPIP solution, wrapped in foil.
- Tube B – 2 cm³ of chloroplast suspension and 8 cm³ of DCPIP solution in light.
- Tube C – 2 cm³ of solution without chloroplasts and 8 cm³ of DCPIP solution in light.
She recorded the colour of the DCPIP in each tube at the start and after the tubes had been left at 25 °C for 20 minutes.
Her results are shown in Table 1.
Table 1
| Tube | Colour of DCPIP at start | Colour of DCPIP after 20 minutes |
|---|---|---|
| A | blue | blue |
| B | blue | colourless |
| C | blue | blue |
Explain why the student set up Tube C.
(2 marks)
Hint
Tube C has light but no chloroplasts. What two things does its result tell you?
Mark Scheme
- To show light does not affect DCPIP (1 mark)
- To show chloroplasts are required (1 mark)
Also accepted / rejected
Ignore: comparison with other tubes.
Tips
- ‘It’s a control’ or ‘for comparison’ isn’t enough: say what Tube C shows, that light alone doesn’t change DCPIP and that chloroplasts are needed
A student mixed a chloroplast suspension from pea leaves with DCPIP solution and left the tube in the light. DCPIP solution is blue when oxidised and colourless when reduced.
After 20 minutes, the DCPIP in the tube had changed from blue to colourless.
Explain this result.
(2 marks)
Hint
DCPIP goes colourless when reduced. What does it accept, and where do those come from in the light-dependent reaction?
Mark Scheme
- Reduction of DCPIP by electrons (1 mark)
- (From) chlorophyll/light dependent reaction (1 mark)
Also accepted / rejected
1. Accept: hydrogen/H for electrons but not protons/hydrogen ions/H⁺ on their own.
2. Accept: from chloroplasts/photosystems/water.
Tips
- DCPIP is reduced by electrons from chlorophyll in the light-dependent reaction, not by protons or reduced NADP
- DCPIP turning colourless is reduction, not oxidation
Scientists tested extracts from several plant species as possible natural herbicides (weed-killers). They assessed each extract by its ability to prevent the decolourisation of DCPIP in illuminated chloroplast suspensions. DCPIP solution is blue when oxidised and colourless when reduced.
They added different concentrations of each extract to the chloroplast suspensions containing DCPIP. They then determined the IC30 for each extract.
The IC30 is the concentration of extract which inhibits the decolourisation of DCPIP by 30%.
Explain the advantage of the scientists using the IC30 in this investigation.
(1 marks)
Hint
Why is using the same 30% point useful when testing lots of extracts?
Mark Scheme
Provides a standard / reference point
OR Can compare different extracts/herbicides
OR Can compare different concentrations of extracts/herbicides (1 mark)
Also accepted / rejected
Accept: decolourises quicker than 100% or saves time waiting for complete decolourisation.
Note: comparisons must be qualified.
Accept: find the most effective extract/herbicide or the most effective concentration.
Accept: answers relating to cost effectiveness.
Tips
- Qualify the comparison: say what can be compared (different extracts or concentrations); ‘for comparison’ alone isn’t enough, and don’t just repeat the stem
Scientists tested extracts from several plant species as possible natural herbicides (weed-killers). They assessed each extract by its ability to prevent the decolourisation of DCPIP in illuminated chloroplast suspensions. DCPIP solution is blue when oxidised and colourless when reduced.
Explain how extracts which inhibit the decolourisation of DCPIP could slow the growth of weeds.
(2 marks)
Hint
If the light-dependent reaction is inhibited, which two products are missing, and which step of the Calvin cycle needs them?
Mark Scheme
Max 2 marks
- Less/no ATP produced (1 mark)
- Less/no reduced NADP produced (1 mark)
- Less/no GP reduced/converted to TP (1 mark)
Also accepted / rejected
2, Accept: less/no NADPH/NADPH₂/NADPH + H
Tips
- Name the products of the light-dependent reaction that won’t be made: ATP and reduced NADP
- Better still, say GP won’t be reduced to TP in the light-independent reaction
1. Accept: osmosis would occur if water potentials were not the same.
1 and 2, Accept: correct reference to osmotic lysis for 2 marks.
2. Accept: chloroplast would burst/lyse/shrivel/shrink if water potentials were not the same.
2. Reject: ‘cell bursts/shrivels’
2. Ignore: damage to chloroplasts on its own is not enough for a mark.
2. Reject: becomes turgid/flaccid.