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Investigating Photosynthesis

Photosynthesis occurs in two separate reactions; the light-dependent stage and the light independent stage. In the light dependent stage the products produced are ATP, NADPH and Oxygen. Oxygen is released through the stomata and the ATP and NADPH are released into the stroma for the light-independent stage, also known as the Calvin cycle. Here Carbon Dioxide is reduced to form various compounds, using RuBP. This reaction also produces NADP and ATP + Pi which is sent back to be used in the light dependent stage.

Method & Equipment
Equipment: • Fresh green spinach, lettuce or cabbage leaves • Scissors • Cold pestle and mortar • Muslin or fine nylon mesh • Filter funnel • Centrifuge and centrifuge tubes • Ice­water­salt bath • DCPIP solution Safety: • DCPIP can stain so avoid skin contact. Method: Isolating the chloroplasts Please note: Keep solutions and apparatus cold during the extraction procedure, steps 1–8, to preserve enzyme activity. The extraction should also be carried out as quickly as possible. 1. Cut three small green spinach, lettuce or cabbage leaves into small pieces with scissors, but discard the tough midribs and leaf stalks. Place in a cold mortar or blender containing 20 cm3 of cold isolation medium (scale up quantities for blender if necessary). 2. Grind vigorously and rapidly (or blend for about 10 s). 3. Place four layers of muslin or nylon in a funnel and wet with cold isolation medium. • Glass rod or Pasteur pipette • Measuring cylinder, 20 cm3 • Beaker, 100 cm3 • Pipettes, 5 cm3 and 1 cm3 • Bench lamp with 100 W bulb • 0.05 M phosphate buffer solution, pH 7.0 • Isolation medium (sucrose and KCl in phosphate buffer)

Place tube 3 in darkness. Results Tube 1 2 3 4 5 Observations/Changes . Re-suspend the pellet with about 2 cm3 of isolation medium. Pour off the liquid (supernatant) into a boiling tube being careful not to lose the pellet. shake the tube and note the time. Note any changes observed in the five tubes.5 – – – – – 0. wring thoroughly into the beaker and add to the tubes.5 – 0.5 0. Centrifuge the tubes for sufficient time to get a small pellet of chloroplasts (10 minutes at high speed 7. using a glass rod. 8. Using the chloroplasts Note: The DCPIP solution should be used at room temperature. Squirting in and out of a Pasteur pipette five or six times gives a uniform suspension. 2 and 4 about 12–15 cm from a bright light (100 W).5 – 0.4. Set up five labelled tubes as follows: Tube 1 2 3 4 5 Leaf extract / Supernatant / Isolation 3 3 cm medium/cm3 cm 0. Place tubes 1. Check that each centrifuge tube contains about the same volume of filtrate. Filter the mixture through the funnel into the beaker and pour the filtrate into pre­ cooled centrifuge tubes supported in an ice­water­salt bath. Gather the edges of the muslin. Store this leaf extract in an ice­water­salt bath and use as soon as possible.5 – – – Distilled water/cm3 – – – 5 – DCPIP solution/cm3 5 5 5 – 5 When the DCPIP is added to the extract. 6. 5.