practical 8

• Dehydrogenase


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

  1. Electrons are accepted by NADP

  2. Dehydrogenase catalyses this reaction
• DCPIP


  Turns from blue to colourless when it is reduced
• Equipment list


  • Leaf sample
  • Isolation solution
  • Ice water bath
  • Distilled water
  • DCPIP
  • Pestle and mortar
  • Test tubes
  • Test tube rack
  • Syringes
  • Pipettes
  • Lamp
  • Timer
  • Tape measure
  • Muslin cloth
  • Funnel
  • Beaker
  • Centrifuge
  • Centrifuge tubes
  • Colorimeter
  • Cuvettes
• Method


  1. Remove stalks from leaf samples

  2. Grind sample using pestle and mortar and place into chilled isolation solution

  3. Filter sample into beaker and suspend in ice water bath

  4. Centrifuge at high speed for 10 minutes to separate chloroplasts into pellet

  5. Remove supernatant and add pellet to fresh isolation medium

  6. Set colorimeter to red filter and zero using chloroplast extract and distilled water

  7. Place test tube 30cm from light source, add DCPIP, and immediately take sample to measure absorbance

  8. Take sample and measure absorbance every 2 minutes for 10 minutes

  9. Repeat for different distances from lamp up to 100cm to vary light intensity
• 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.
• Hazards


  • DCPIP (irritant to skin and eyes, may cause staining)
  • Biohazard (allergies, soil bacteria, contamination)
  • Lamps (temporary damage to eyes)
  • Electrical appliances (liquids near)
• Safety precautions


  • Wear eye protection, wash from skin/eyes immediately using cold water
  • Wash hands after use, seek assistance
  • Do not look directly at lamp, wait for after image to disappear, seek appropriate assistance if needed
  • Do not touch lamp/wires with wet hands, keep liquids away from lamp/wires, seek assistance
• As the light intensity decreases


  The rate of photosynthesis also decreases
• Lowered 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 the chlorophyll

  DCPIP accepts less electrons
• DCPIP is blue


  Absorbance is higher
• Higher rate of decrease in absorbance

  Indicates the dehydrogenase enzyme is highly active