8. Dehydrogenase Activity in Chloroplasts (rate of photosynthesis) Flashcards
Investigation into the effect of a named factor on the rate of dehydrogenase activity in extracts of chloroplasts
What is the function of dehydrogenase in chloroplasts?
It catalyses the acceptance of electrons by NADP in the light dependent reactions.
What is the purpose of DCPIP?
It is a redox indicator dye and acts as an alternate electron acceptor instead of NADP.
It turns from blue to colourless when reduced.
Why is the plant extract chilled in an ice-water bath?
To lower the activity of enzymes to prevent them from breaking down the chloroplasts.
How is the control set up?
Fill a cuvette with chloroplast extract and distilled water.
How is light intensity controlled?
Adjust the distance of the lamp from the set up.
Perform the practical in a dark room so that the only light source is the lamp (more reliable).
The sample should not be put too close to the lamp as temperature may affect the results.
How is the chloroplast extract prepared?
- Remove stalks from leaf samples. 2. Grind sample using a pestle and mortar and place into a chilled isolation solution.
- Use a muslin cloth and funnel to filter the sample into a beaker.
- Suspend the beaker in an ice water bath to keep sample chilled.
- Transfer to centrifuge tubes and centrifuge at high speed for 10 minutes. This will separate chloroplasts into the pellet.
- Remove supernatant and add pellet to the fresh isolation medium. Store isolation solution on ice.
What is the function of the muslin cloth?
To filter out any debris in the ground leaf mixture but allowing chloroplasts to pass through.
Why are the stalks of leaves removed before grinding?
The stalks do not contain many chloroplasts.
Outline the procedure of investigating the effect of light intensity, after chloroplast extract has been obtained.
- Set the colorimeter to the red filter. Zero using a cuvette containing chloroplast extract and distilled water (control).
- Place test tube in the rack 30cm from light source and add DCPIP. Immediately take a sample and add to cuvette. Measure the absorbance of the sample.
- Take a sample and measure its absorbance every 2 minutes for 10 minutes.
- Repeat for different distances from lamp up to 100 cm.
Risks & Precautions
DCPIP is an Irritant to skin and eyes; may cause staining.
Biohazard: Allergies; soil bacteria; contamination
Lamps: Temporary damage to eyes
How do you plot a graph of results of rate of photosynthesis?
Plot a graph of DCPIP absorbance against time for each distance from the light.
Conclusion: effect of light intensity on rate of photosynthesis?
● As the light intensity decreases, the rate of photosynthesis decreases. This is because the lowered light intensity will slow the rate of photoionisation of the chlorophyll pigment, so the overall rate of the light dependent reaction will be slower.
● This means that less electrons are released by the chlorophyll, hence the DCPIP accepts less electrons. This means that it will take longer to turn from blue to colourless.
● When the DCPIP is blue, the absorbance is higher. The rate at which the absorbance decreases can therefore be used to determine the activity of the dehydrogenase enzyme.
● A higher rate of decrease, shown by a steep gradient on the graph, indicates that the dehydrogenase is highly active.
