Dehydrogenase Activity in Chloroplasts (AQA A-Level Biology): Revision Notes
Dehydrogenase Activity in Chloroplasts
Purpose and principle
This required practical investigates how light intensity affects the rate of dehydrogenase activity in chloroplast extracts. Dehydrogenase enzymes play an essential role in the light-dependent reactions of photosynthesis, where they facilitate the acceptance of electrons by NADP.
The practical uses DCPIP (2,6-dichlorophenolindophenol) as a redox indicator dye. When dehydrogenase is active, electrons are transferred to DCPIP instead of NADP, causing the dye to change from blue to colourless as it becomes reduced. By measuring this colour change using a colorimeter, we can determine the enzyme's activity level.
The key principle behind this investigation is that DCPIP acts as an artificial electron acceptor, competing with NADP for electrons from the light-dependent reactions. The rate of DCPIP reduction (colour change) directly reflects the activity of dehydrogenase enzymes under different light conditions.
Apparatus and materials
The practical requires standard laboratory equipment including:
- Fresh leaf samples and isolation solution for preparing chloroplast extracts
- DCPIP solution as the redox indicator
- Pestle and mortar for tissue disruption
- Centrifuge and centrifuge tubes for separating chloroplasts
- Colorimeter with red philtre and cuvettes for measuring absorbance
- Test tubes, syringes, and pipettes for handling solutions
- Light source (lamp) and tape measure for controlling light intensity
- Ice water bath to maintain sample temperature
- Muslin cloth and funnel for filtration
All chloroplast preparations must be kept on ice throughout the experiment to prevent enzyme denaturation and maintain chloroplast integrity. The isolation solution should also be pre-chilled before use.
Method
The investigation examines light intensity as the independent variable by varying the distance between the light source and the sample.
Sample preparation
Remove stalks from fresh leaf samples and grind the tissue using a pestle and mortar with chilled isolation solution. This solution helps maintain chloroplast integrity during extraction.
Philtre the mixture through muslin cloth into a beaker, then transfer to centrifuge tubes. Centrifuge at high speed for 10 minutes to separate the chloroplasts, which form a pellet at the bottom. Remove the supernatant and resuspend the pellet in fresh isolation solution, keeping the extract on ice throughout.
Sample Preparation Steps:
Step 1: Grind 5g of fresh spinach leaves with 20ml chilled isolation solution
Step 2: Philtre through muslin cloth to remove large debris
Step 3: Centrifuge at 3000rpm for 10 minutes
Step 4: Discard supernatant and resuspend pellet in 10ml fresh isolation solution
Step 5: Keep on ice until ready for use
Measurement procedure
Set up the colorimeter using a red philtre and zero the instrument with a cuvette containing chloroplast extract and distilled water. Position the test tube containing chloroplast extract and DCPIP at 30cm from the light source initially.
Add DCPIP to the sample and immediately measure the absorbance using the colorimeter. Record further absorbance readings every 2 minutes for 10 minutes to track the colour change over time.
Repeat the measurements at different distances from the lamp (up to 100cm) to vary the light intensity. The experiment should be conducted in a darkened room to ensure accurate light control, avoiding placing samples too close to prevent temperature effects.
Light intensity follows the inverse square law, so doubling the distance from the light source reduces intensity to one-quarter of the original value. This relationship allows you to calculate relative light intensities at different distances.
Safety considerations
Several hazards require appropriate precautions:
- Chemical Hazards: DCPIP can irritate skin and eyes, potentially causing staining. Wear eye protection throughout and wash immediately with cold water if contact occurs.
- Biological Hazards: The biological material presents biohazard risks including allergies, soil bacteria, and contamination. Wash hands thoroughly after handling samples and seek assistance if allergic reactions develop.
- Electrical Safety: Electrical equipment poses risks from liquids near appliances. Keep all solutions away from electrical connections and avoid touching equipment with wet hands.
- Light sources may cause temporary eye damage, so avoid looking directly at lamps and seek assistance if afterimages persist.
Data collection and analysis
Plot graphs showing absorbance against time for each distance from the light source. The rate of absorbance decrease indicates dehydrogenase activity levels.
As light intensity decreases with greater distance, the rate of photosynthesis also decreases. This occurs because lower light intensity reduces the rate of photoionisation of chlorophyll pigments, slowing the overall light-dependent reactions.
Consequently, fewer electrons are released by chlorophyll, meaning DCPIP accepts fewer electrons and takes longer to change from blue to colourless. When DCPIP remains blue, the absorbance remains higher for longer periods.
A steeper gradient on the absorbance-time graph indicates higher dehydrogenase activity, whilst a shallower gradient suggests lower activity levels under reduced light conditions.
Graph Interpretation:
At 30cm from light source:
- Initial absorbance: 1.2
- Final absorbance: 0.3
- Rate of decrease: 0.15 absorbance units per minute
At 80cm from light source:
- Initial absorbance: 1.2
- Final absorbance: 0.8
- Rate of decrease: 0.07 absorbance units per minute
The steeper gradient at 30cm indicates higher dehydrogenase activity due to greater light intensity.
Links to theory
This practical demonstrates the relationship between light intensity and the light-dependent stage of photosynthesis. It specifically examines how electron transport chains function within chloroplasts and how enzyme activity can be measured using indicator dyes.
The results connect to broader understanding of limiting factors in photosynthesis and how environmental conditions affect metabolic processes in plant cells.
This investigation links directly to the concept of limiting factors in photosynthesis. When light intensity is the limiting factor, increasing it will increase the rate of photosynthesis until another factor (such as temperature or CO₂ concentration) becomes limiting.
Remember!
Key Points to Remember:
- Dehydrogenase catalyses electron acceptance by NADP in the light-dependent reactions of photosynthesis
- DCPIP acts as an artificial electron acceptor, changing from blue to colourless when reduced
- Light intensity affects the rate of photoionisation, which influences dehydrogenase activity
- Colorimeter measurements track the rate of DCPIP reduction as an indicator of enzyme activity
- Steeper gradients on absorbance-time graphs indicate higher dehydrogenase activity levels