RP8 Investigation into the effect of a named factor on the rate of dehydrogenase activity in extracts of chloroplasts

Describe the role of the enzyme dehydrogenase in photosynthesis. (2 marks)

- Catalyses the reduction of NADP in the light-dependent reaction.

- NADP accepts (gains) electrons from photoionisation of chlorophyll or from photolysis of water.

Describe how the rate of dehydrogenase activity in extracts of chloroplasts can be measured. (5 marks)

1. Extract chloroplasts from a leaf sample using ultracentrifugation.

2. Set up the experimental test tubes.

3. Shine light on the tubes and time how long it takes for DCPIP in the experimental tube to turn from blue (oxidised) to colourless (reduced).

4. Comparing to a colour standard (tube C) to identify the endpoint.

5. Calculate the rate of dehydrogenase activity (s⁻¹) as 1 ÷ time taken.

Describe how you would set up the test tubes for investigating the rate of dehydrogenase activity in extracts of chloroplasts. (4 marks)

- Control 1 = set volume of DCPIP, water, and isolation medium, with chloroplasts present, covered in foil to block light.

- Control 2 = set volume of DCPIP, water, and isolation medium, without chloroplasts.

- Standard = set volume of water and chloroplasts in isolation medium, without DCPIP.

- Experiment = set volume of DCPIP, water, and isolation medium with chloroplasts present.

Explain the purpose of control 1 (tube A) in the investigation. (2 marks)

- Demonstrates that light is necessary for DCPIP to decolourise.

- Demonstrates that chloroplasts alone do not cause DCPIP to decolourise.

Explain why DCPIP in control 1 (tube A) remains blue. (2 marks)

- Absence of light prevents photoionisation of chlorophyll.

- Therefore, no electrons are released to reduce DCPIP.

Explain the purpose of control 2 (tube B) in the investigation. (2 marks)

- Demonstrates that chloroplasts are required for DCPIP to decolourise.

- Demonstrates that light alone does not cause DCPIP to decolourise.

Describe why DCPIP changes from blue to colourless in the experiment. (2 marks)

- DCPIP is a redox indicator that becomes reduced when it gains electrons.

- Electrons are released during photoionisation of chlorophyll.

State three variables that should be controlled when measuring dehydrogenase activity. (3 marks)

- Source of chloroplasts.

- Volume of chloroplast suspension.

- Volume or concentration of DCPIP.

Explain one limitation of this method and how it could be improved. (4 marks)

- Decolourisation end point is subjective.

- Use a colorimeter for objective measurement.

- Measure absorbance of the sample at regular time intervals.

- Calibrate the colorimeter using a colour standard.