5.6.10 PRACTICAL: Measuring the rate of the LDS of photosynthesis

Question 1

Describe the LDS and how this practical can be carried out.

Answer 1

• The light-dependent reactions of photosynthesis take place in the thylakoid membrane and involve the release of high-energy electrons from chlorophyll a molecules
• These electrons are picked up by the electron acceptor NADP in a reaction catalysed by the enzyme dehydrogenase
• However, if a redox indicator (such as DCPIP or methylene blue) is present, the indicator takes up the electrons instead of NADP

This causes the indicator to change colour
- DCPIP: oxidised (blue) → accepts electrons → reduced (colourless)

• Methylene blue: oxidised (blue) → accepts electrons → reduced (colourless)
• The colour of the reduced solution may appear green because chlorophyll produces a green colour

The rate at which the redox indicator changes colour from its oxidised (blue) state to its reduced (colourless) state can be used as a measure of the rate of dehydrogenase activity and therefore, the rate of the light-dependent stage of photosynthesis
- When light is at a higher intensity, or at more preferable light wavelengths, the rate of photoactivation of electrons is faster, therefore the rate of reduction of the indicator is faster

Question 2

What apparatus is needed?

Answer 2

Leaves
Isolation medium
Pestel and mortar
Lamp
Test tubes
Stopwatch
Aluminium Foil
DCPIP or methylene blue indicator
Buffer solution

Question 3

Describe the method of measuring light as a limiting factor.

Answer 3

Leaves are crushed in a liquid known as an isolation medium
- This produces a concentrated leaf extract that contains a suspension of intact and functional chloroplasts
- The medium must have the same water potential as the leaf cells so the chloroplasts don’t shrivel or burst and contain a buffer to keep the pH constant
- The medium should also be ice-cold (to avoid damaging the chloroplasts and to maintain membrane structure)

The experiment should be set up in a dark room so that the light source and intensity can be controlled
- The room should be at an adequate temperate for photosynthesis and maintained throughout, as should carbon dioxide concentration

Small tubes are set up with different intensities, or different colours (wavelengths) of light shining on them
- If different intensities of light are used, they must all be of the same wavelength (same colour of light) – light intensity is altered by changing the distance between the lamp and the test tube
- If different wavelengths of light are used, they must all be of the same light intensity – the lamp should be the same distance in all experiments

• DCPIP or methylene blue indicator is added to each tube, as well as a small volume of the leaf extract
• A control that is not exposed to light (wrapped in aluminium foil) should also be set up to ensure the affect on colour is due to the light

The time taken for the redox indicator to go colourless (or green, as the chlorophyll may also colour the solution) is recorded
- This is a measure of the rate of photosynthesis

Question 4

Describe the results.

Answer 4

• A graph should be plotted of absorbance against time for each distance from the light

As the light intensity decreases, the rate of photosynthesis also 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.

Question 5

What are the limitations?

Answer 5

• This experiment is not measuring the rate of dehydrogenase activity directly (through measuring the rate of substrate use or product made) but is instead predicting what the rate would be by measuring the rate of electron transfer from the photosystems

The concentration of DCPIP will depend on the number of chloroplasts in a sample and therefore the number of light-dependent electron transport chains
- It is therefore important to control the amount of leaf used to produce the chloroplast sample and also how much time is spent crushing the leaf to release the chloroplast
- It is also a good idea to measure a specific wavelength absorption by each sample on the colorimeter before and after the experiment so you can get a more accurate change in oxidised DCPIP concentration
- Results should also be repeated and the mean value calculated

• The time taken to go colourless is subjective to each person observing and therefore one person should be assigned the task of deciding when this is