Required Practical 8 Dehydrogenase activity in chloroplasts
Joe Wolfensohn
Teacher
Contents
Recall Questions
This topic requires prior knowledge of the light dependent reaction. You can test your knowledge on this below.
Where does the light-dependent reaction take place?
On the thylakoid membranes within the chloroplasts.
What happens to the electrons excited from chlorophyll during the light-dependent reaction?
They are transferred along the electron transport chain, releasing energy to generate ATP via chemiosmosis.
What is the final electron acceptor in the light-dependent reaction?
NADP, which forms reduced NADP (NADPH) after accepting electrons and a proton.
Topic Explainer Video
Check out this @JoeDoesBiology video that explains dehydrogenase activity in chloroplasts or read the full notes below. Once you've gone through the whole note, try out the practice questions!
Purpose and Principle Behind the Practical
Purpose of the Practical
This practical investigates dehydrogenase enzyme activity in chloroplasts during the light-dependent reaction of photosynthesis.
Dehydrogenase enzymes are enzymes that transfer hydrogen atoms (and electrons) from one molecule to another.
This experiment uses a redox indicator, DCPIP, which changes colour when reduced (gains electrons), allowing us to monitor electron transfer in chloroplasts.
Principle Behind the Practical
-
In the LDR, electrons released from chlorophyll (via photoionisation) are transferred through the electron transport chain.
-
Normally, these electrons reduce NADP to NADPH.
-
In this experiment, DCPIP (blue) is used as an artificial electron acceptor instead of NADP.
-
When DCPIP is reduced, it becomes colourless.
- The faster the colour change, the faster the rate of dehydrogenase activity, and thus electron transfer.
Simplified Method
-
Isolate chloroplasts from fresh green leaves (e.g., spinach) using ice-cold buffer and a centrifuge.
-
Set up three test tubes with the following conditions:
|
Tube |
Contents |
Light Condition |
|---|---|---|
|
A |
DCPIP + isolation buffer (no chloroplasts) |
Light |
|
B |
DCPIP + chloroplast suspension |
Light |
|
C |
DCPIP + chloroplast suspension |
Dark (wrapped in foil or kept in a dark cupboard) |
-
Control for volume: Ensure each tube contains equal volumes (e.g., 1 cm³ DCPIP + 5 cm³ buffer/chloroplast suspension).
-
Place Tube A and B under a light source, and Tube C in a completely dark environment (e.g. wrapped in foil or inside a closed drawer).
-
Record the time taken for DCPIP to turn from blue to colourless
Expected Results and Explanation
|
Tube |
Expected Observation |
Explanation |
|---|---|---|
|
A (no chloroplasts) |
No colour change – DCPIP remains blue |
No chloroplasts = no electron donors = no reduction of DCPIP |
|
B (light + chloroplasts) |
DCPIP decolourises rapidly (blue → colourless) |
Light excites electrons in chlorophyll → electrons reduce DCPIP |
|
C (dark + chloroplasts) |
Little or no colour change |
No light = no photoionisation of chlorophyll = no electron transfer to DCPIP |
Extending the experiment
The effect of ammonium hydroxide on the time taken for chloroplasts to decolourise DCPIP.
What is Ammonium Hydroxide (NH₄OH)?
-
It’s a weak base that increases the pH of the solution.
-
It can disrupt thylakoid membranes.
-
In high concentrations, it may denature proteins, including enzymes and structural proteins within chloroplast membranes.
-
It acts in the same way as many weed killers.
You could add an extra test tube to investigate the effect of ammonium hydroxide on electron transfer:
|
Tube |
Contents |
Light Condition |
|---|---|---|
|
D |
Chloroplasts + DCPIP + Ammonium hydroxide |
Light |
Likely Observations:
-
DCPIP decolourises more slowly or not at all compared to the normal light condition.
Why This Happens – Explanation Linked to the LDR:
|
Effect |
Mechanism |
|---|---|
|
Effect on electron transport |
High pH or membrane disruption may affect the structure of photosystems or electron carriers, reducing the flow of electrons from chlorophyll. |
|
Reduction in DCPIP |
If fewer electrons are passed down the ETC, less DCPIP is reduced, so the colour change slows or stops. |
Key Terms
-
DCPIP (2,6-dichlorophenol-indophenol): A redox indicator that acts as an artificial electron acceptor; it changes from blue to colourless when reduced.
-
Dehydrogenase: General term for enzymes that transfer hydrogen atoms (and electrons) from one molecule to another.
-
Photoionisation: The process where chlorophyll absorbs light and loses electrons.
-
Electron Transport Chain (ETC): A series of proteins in the thylakoid membrane that transfer electrons, releasing energy.
No answer provided.
Exam Tip
Control experiments are important and allow for comparison. Here tube A and tube C both acted as controls.
Tube A shows that the colour change of DCPIP in tube B is due to the presence of chloroplasts/ chlorophyll.
Tube C shows that light energy is required for the transfer of electrons and the reduction of DCPIP.
When comparing tube B to these control tubes we can then conclude that DCPIP changed from blue to colourless due to its reduction by electrons and that these electrons came from chlorophyll during the light dependent reaction.
No answer provided.
In an experiment a student evaluated the effectiveness of different chemicals as weed-killers that prevent the decolourisation of DCPIP in chloroplast suspensions.
Explain how chemicals which inhibit the decolourisation of DCPIP could slow the growth of weeds. (3 marks)
1. Less / no ATP produced
2. Less / no reduced NADP produced
3. Less / no GP reduced to triose phosphate
Practice Question
Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!