Required practical 8
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Describe the role of the enzyme dehydrogenase in photosynthesis
● Catalyses the reduction of NADP in the light-dependent reaction ○ NADP accepts (gains) electrons from photoionisation of chlorophyll / photolysis of water
Describe how rate of dehydrogenase activity in extracts of chloroplasts can be measured
• Extract chloroplasts from a leaf sample using cell fractionation • 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 • Shine light on test tubes and time how long to 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 • Rate of dehydrogenase activity (s-1) = 1 / time taken
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 with foil)
● Shows light is required for DCPIP to decolourise ● Shows that chloroplasts alone do not cause DCPIP to decolourise
Explain why DCPIP in control tube with foil stays blue.
● No light so 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 is a redox indicator / DCPIP gets 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 colorimeter ● Measure light absorbance of sample at set time intervals ● Zero colorimeter using the colour standard