Investigating Photosynthesis

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Investigating Dehydrogenase Activity in Chloroplasts

During the light-dependent reaction, NADP is reduced to NADPH. A dehydrogenase enzyme catalyses this reaction. This experiment will monitor dehydrogenase activity by using DCPIP, a redox indicator.

DCPIP

In this investigation, a blue dye (DCPIP) is used to monitor the rate of dehydrogenase activity. DCPIP is a redox indicator.
This means that it is blue in the oxidised state and colourless in its reduced state.
When electrons are released by the chlorophyll, DCPIP will change from blue to colourless.

1) Extracting chloroplasts

Put 50 cm³ of isolation medium into a beaker.
Tear eight spinach leaves into small pieces and put the pieces into the isolation medium in the beaker.
- Do NOT put pieces of the midrib or the leaf stalk into the beaker.
Half fill a large beaker with ice and place a small beaker on top of the ice.
When carrying out this step, all solutions and apparatus should be kept as cold as possible and the extraction should be carried out as quickly as possible.

2) Suspending chloroplasts

Put 3 layers of muslin over the top of the filter funnel and wet with the isolation medium. Rest the filter funnel in the small beaker on the ice.
Pour the spinach and isolation medium into the blender and blend for 15 seconds. Pour the blended mixture back into the beaker.
Pour your blended mixture through the muslin in the filter funnel. Carefully squeeze the muslin to assist the filtering process.
Label this filtrate which is in the small beaker on ice as ‘chloroplast suspension’.

4) Set up tubes A and B

Label five test tubes A, B, C, X and Y and stand them in the large beaker. Put the lamp about 10 cm away so that all tubes are illuminated. Set up tubes A and B as follows:
- Tube A - 5 cm³ DCPIP solution + 1 cm³ water + 1 cm³ chloroplast suspension. Immediately wrap the tube in aluminium foil to exclude light.
- Tube B - 5 cm³ DCPIP solution + 1 cm³ water + 1 cm³ isolation medium.
Tubes A and B are control experiments. Leave both tubes until the end of your investigation.

5) Set up tube C

Set up tube C as follows:
- Tube C - 6 cm³ water + 1 cm³ chloroplast suspension.
Tube C is for you to use as a standard to help you to determine when any colour change is complete.

6) Set up tube X

Set up tube X as follows:
- Tube X - 5 cm³ DCPIP solution + 1 cm³ water in the tube.
Add 1 cm³ chloroplast suspension to tube X, quickly mix the contents and start the timer.
Record in seconds how long it takes for the contents of tube X to change colour from blue-green to green. Use tube C to help you determine when the colour change is complete.
- Repeat this step four more times.

7) Set up tube Y

Set up tube Y as follows:
- Tube Y - 5 cm³ DCPIP solution + 1 cm³ ammonium hydroxide.
Add 1 cm³ chloroplast suspension to tube Y, quickly mix the contents and start the timer.
Record in seconds how long it takes for the contents of tube Y to change colour from blue-green to green. Use tube C to help you determine when the colour change is complete.
- Repeat this step four more times.

8) Record the results

Record your data in a suitable table.
At the end of your investigation, record the colour of the mixtures in tubes A and B.

The Hill Reaction

The Hill reaction was named after Robert Hill, who designed this experiment to understand more about the process of photosynthesis. It investigates the light-dependent reaction in photosynthesis.

The Hill Reaction

The Hill reaction follows these generalised steps:
- Extract chloroplasts from a plant.
- Suspend chloroplasts in solution.
- Add DCPIP (an electron acceptor in place of NADP), which is used to observe whether redox reactions are taking place, to the treatment solutions and a control solution.
- Expose chloroplast solutions to light and record any colour changes of DCPIP.

DCPIP

DCPIP is an electron acceptor which acts as a substitute for NADP in this reaction.
DCPIP is blue when oxidised, and colourless when reduced.
If DCPIP turns from blue to colourless, this means it is accepting electrons and a redox reaction is taking place.

Possible reactions to investigate

The Hill reaction can be used to investigate the effect of different variables on photosynthesis.
Solutions can be heated to different temperatures to investigate how temperature affects rate of photosynthesis.
Solutions can be exposed to light or dark to see how light intensity affects rate of photosynthesis.
Solutions can be made up to different pH levels to investigate effect of pH on rate of photosynthesis.
The speed at which DCPIP changes from blue to colourless is inversely related to rate of photosynthesis.

Important findings

Hill found that when isolated chloroplasts were exposed to light, the added DCPIP turned from blue to colourless, but not when the chloroplasts were kept in the dark.
This was an important finding because it showed that photosynthesis depends at least partly on light, and that redox reactions were happening.
The reaction also showed that light was needed for oxygen to be produced, and that production of oxygen happens in a different step to fixation of carbon dioxide.