RPQ 8 - investigating the activity of dehydrogenase

• in photosystem 1 during the LDR stage of photosynth, NADP acts as an electron acceptor and is reduced:

  • the reaction is catalysed by a dehydrogenase enzyme
  • the activity of this enzyme can be investigated by adding a redox indicator dye to extracts of chloroplats
• • like NADP, the dye acts as an electron acceptor and gets reduced by the dehydrogenase in the chloroplasts
  • as the dye gets reduced - colour change
  • e.g. the dye DCPIP changes from blue to colourless when it is reduced
• • you can measure the rate of the dehydrogenase activity by measuring the rate at which DCPIP loses its blue colour
  • need a colorimeter - measures how much light a solution absorbs when a light source is shone directly through it
  • a coloured solution absorbs more light than a colourless one
• this example shows you how to investigate the effect of light intensity on dehydrogenase activity in extracts of chloroplasts:

  • it uses a bench lamp as a light source and involves placing tubes of chloroplasts mixed with DCPIP at a range of different distances from the light source/lamp
  • need to choose distances going to investigate e.g. 15cm, 30cm, 45cm before you start
• 1. cut a few leaves (spinach works well) into pieces and remove any tough stalks
  2. using a pestle and mortar grind up the leaf pieces with some chilled isolation solution (solution of sucrose, potassium chloride and phosphate buffer at pH 7). filter the liquid you make into a beaker through a funnel lined with muslin cloth
  3. transfer the liquid to centrifuge tubes and centrifuge them at high speed for 10 min - make the chloroplasts gather at the bottom of the tube in a pellet
• 4. get rid of the liquid from the top of the tubes leaving the pellets in the bottom

  5. resuspend the pellets in fresh, chilled isolation solution - this is the chloroplast extract store it on ice for the rest of the experiment

  6. set up a colorimeter with a red filter and zero it using a cuvette (cuboid shaped vessel used in colorimeters) containing chloroplast extract and distilled water
• 7. set up a test tube rack at a set at a set distance from the bench lamp switch the lamp on

  8. put a test tube in the rack, add a set volume of chloroplast extract to the tube and a set volume of DCPIP mix the contents of the tube together

  9. immediately take a sample of the mixture from the tube and add it to a clean cuvette. then place the cuvette in your colorimeter and record the absorbance - do it evert 2 mins for the next 10 mins
• 10. repeat steps 7-9 for each distance under investigation

  11. you should also check whether the absorbance changed at each distance in two negative control tubes

  • the first should only contain DCPIP and chilled isolation solution (no chloroplast extract)
  • the second should contain both DCPIP and chloroplast extract but it should be wrapped in tin foil so no light reaches the contents of the tube
  • no change in absorbance should be seen in these 2 controls
• if dehydrogenase activity is taking place - the absorbance will decrease as the DCPIP gets reduced and loses its blue colour

  the faster the absorbance decreases, the faster the rate of dehydrogenase activity
• can plot a graph of absorbance against time for each distance from the light source - then compare your results to determine how light intensity affects the rate of the dehydrogenase enzyme
• a centrifuge is a machine that spins samples really quickly - the resulting force separates out the components of your sample

  always make sure your centrifuge is balanced by placing tubes of equal weight opposite each other
• can use a similar method to investigate the effects of other factors on the activity of dehydrogenase in chloroplasts e.g. temp and photosynthetic inhibitors
• the first neg control tube should show that the chloroplast extract is needed to make DCPIP change colour
• the second neg control tube should show that light is needed to make DCPIP change colour