RP11

• Dehydrogenase


  An enzyme found in plant chloroplasts that is crucial to the light dependent stage of photosynthesis
• Light dependent stage of photosynthesis

  Electrons are accepted by NADP
• Hill reaction


  The reaction where dehydrogenase catalyses the acceptance of electrons by NADP, discovered in 1938 by Robin Hill
• DCPIP


  A redox indicator dye that turns from blue to colourless when reduced
• Isolating chloroplasts


  1. Remove stalks from leaf samples

  2. Cut into small sections

  3. Grind sample using pestle and mortar

  4. Place into chilled isolation solution

  5. Filter sample into beaker

  6. Suspend beaker in ice water bath

  7. Centrifuge at high speed for 10 minutes

  8. Remove supernatant and add pellet to fresh isolation medium

  9. Store isolation solution on ice
• Measuring chloroplast activity


  1. Set colorimeter to red filter

  2. Zero using chloroplast extract and distilled water

  3. Place test tube 30cm from light source and add DCPIP

  4. Immediately take sample and add to cuvette

  5. Measure absorbance every 2 minutes for 10 minutes

  6. Repeat for different distances up to 100cm from lamp
• This experiment should be done in a darkened room to make results more reliable
• The sample should not be put too close to the lamp as temperature may affect results
• As light intensity decreases


  The rate of photosynthesis also decreases
• Lower light intensity


  Slows the rate of photoionisation of the chlorophyll pigment, so the overall rate of the light dependent reaction will be slower
• Less electrons released by chlorophyll

  DCPIP accepts less electrons, so it takes longer to turn from blue to colourless
• DCPIP is blue


  Absorbance is higher
• Rate of decrease in absorbance

  Can be used to determine the activity of the dehydrogenase enzyme
• Higher rate of decrease in absorbance

  • Indicates that the dehydrogenase is highly active