Hill rxn - Hill Reaction of Photosynthesis In this...

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Hill Reaction of Photosynthesis In this experiment you will be provided with isolated chloroplast or thylakoids capable of doing some of the parts of photosynthesis in vitro (i.e., in glass, or in the test tube). The Hill reaction will be measured by the reduction of an artificial electron acceptor, 2,6 di-chlorophenol- indo-phenol ( DCIP ). As electrons are taken from water by photosynthesis they are grabbed by DCIP. This causes DCIP (a blue dye) to be reduced (gain electrons) to its colourless form, DCIPH 2 . l i g h t DCIP + H 2 O ----------------------------------> DCIPH 2 + ½ O 2 (blue) chloroplasts/thylakoids (colourless) There is no DCIP in normal chloroplasts. It is added here because it loves electrons and can get them easily from excited chlorophyll-a. DCIP is also very useful because it indicates (by the change from blue to clear) when it has been reduced. We can monitor this colour loss with a simple spectrophotometer. In fact, this is easier than trying to measure the oxygen produced by the splitting of water as the electrons and protons are pulled out of water. The suspensions of chloroplasts or thylakoids have been prepared for you by the technician according to The Isolation of Chloroplasts or Thylakoids below. Please be sure that you understand how the isolation process works so that you can understand what you are using. For this to work you will want to get intact membrane-bound chloroplasts or intact membrane-bound thylakoids. You cannot proceed with just extracted chlorophyll a for this reaction to work. You will receive a suspension of chloroplasts or thylakoids in 20 mL of 0.5M sucrose solution which has been stored on ice as in step 2(g) below. The Isolation of Chloroplasts or Thylakoids 1. Pre-cool a beaker, 12 centrifuge tubes and 6 or 8 Spectronic 20 test tubes in an ice bath. 2. Obtain a homogenate of spinach leaves using the following procedure: a. Blend 45 grams of washed, chilled spinach (or Swiss chard) leaf blades (remove midribs and petioles) with 150 mL of an ice-cold 0.5M aqueous sucrose solution in a blender for 30 seconds at top speed.
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This note was uploaded on 01/25/2012 for the course ECON 120 taught by Professor Dr.hill during the Three '11 term at Australian National University.

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Hill rxn - Hill Reaction of Photosynthesis In this...

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