Required Practical 8

• Describe the role of the enzyme dehydrogenase in photosynthesis:

  • Catalyses the reduction of NADP in the light dependant phase
  • NADP accepts electrons from photoionisation of chlorophyll / photolysis of water
• What is the first step in measuring the rate of dehydrogenase activity in chloroplast extracts?

  Extract chloroplasts from a leaf sample using centrifugation
• What are the components of the test tubes set up for measuring dehydrogenase activity?

  • A = Control 1: DCPIP, water, chloroplasts in isolation medium (covered in foil)
  • B = Control 2: DCPIP, water, isolation medium (without chloroplasts)
  • C = Standard: water, chloroplasts in isolation medium (without DCPIP)
  • D = Experiment: DCPIP, water, chloroplasts in isolation medium
• Why is tube A covered in foil during the experiment?


  To block light from affecting the reaction
• What should be observed in tubes A and B during the experiment?

  There should be no change in color
• How is the endpoint of the experiment identified?


  By comparing the color change in tube D to the color standard in tube C
• What is the formula to calculate the rate of dehydrogenase activity?

  Rate of dehydrogenation activity (s-1) = 1/time taken
• Once test tubes have been set up what happens next?

  Shine light on test tubes and time how long it takes for DCPIP to turn from blue (oxidised) to colourless (reduced) in tube D
• Give examples of variables that could be controlled:

  • Source of chloroplasts
  • Volume of chloroplast suspension
  • Volume / concentration of DCPIP
• Explain the purpose of control 1 (tube A):

  • Shows light is required for DCPIP to decolourise
  • Shows that chloroplasts alone do not cause DCPIP to decolourise
• Explain why DCPIP in control 1 stays blue:

  • No light = no photoionisation of chlorophyll
  • So no electrons released to reduce DCPIP
• Explain the purpose of control 2 (tube B):

  • Shows chloroplasts are required for DCPIP to decolourise
  • Shows that light alone does not cause DCPIP to decolourise
• Explain why DCPIP changes from blue to colourless:

  • DCPIP acts as a final electron acceptor (redox indicator) so is reduced by electrons
  • From photoionisation of chlorophyll
• Suggest a limitation with the method and how the experiment could be modified to overcome this:

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