Required Practical 8

Question 1

Describe the role of the enzyme dehydrogenase in photosynthesis.

Answer 1

• Catalyses the reduction of NADP in the light-dependant reaction.
  
  • NADP accepts (gains) electrons from photoionisation of chlorophyll/photolysis of water.

Question 2

Describe how rate of dehydrogenase activity in extracts of chloroplasts can be measured.

Answer 2

1. Extract chloroplasts from leaf sample using the method in ‘2.1.3 Methods of studying cells’.
2. Set up test tubes as follows:
   
   • A. Control 1 - set volume of DCPIP (redox indicator dye, electron acceptor), water and chloroplasts in isolation medium, covered in foil to block light.
   • B. Control 2 - set volume of DCPIP, water and isolation medium without chloroplasts.
   • C. Standard - set volume of water and chloroplasts in isolation medium, without DCPIP.
   • D. Experiment - set volume of DCPIP, water and chloroplasts in isolation medium.
3. Shine light on test tubes and time how long it takes for DCPIP to turn from blue (oxidised) to colourless (reduced) in tube D (tube A and B should show no change).
   
   • Compare to a colour standard (tube C) to identify end point.
4. Rate of dehydrogenase activity = 1 / time taken.

Question 3

Give examples of variables that could be controlled.

Answer 3

• Source of chloroplasts.
• Volume of chloroplast suspension.
• Volume / concentration of DCPIP.

Question 4

Explain the purpose of control 1 (tube A).

Answer 4

• Shows light is required for DCPIP to decolourise.
• Shows that chloroplasts alone do not cause DCPIP to decolourise.

Question 5

Explain why DCPIP in control 1 stays blue.

Answer 5

• No light so no photoionisation of chlorophyll.
• So no electrons released to reduce DCPIP.

Question 6

Explain the purpose of control 2 (tube B).

Answer 6

• Shows chloroplasts are required for DCPIP to decolourise.
• Shows that light alone does not cause DCPIP to decolourise.

Question 7

Explain why DCPIP changes from blue to colourless.

Answer 7

• DCPIP is a redox indicator / DCPIP gets reduced by electrons.
• From photoionisation of chlorophyll.

Question 8

Suggest a limitation with the method and how the experiment could be modified to overcome this.

Answer 8

• End point (colour change) is subjective.
• Use a colorimeter.
• Measure light absorbance of sample at set time intervals.
• Zero colourimeter using the colour standard.