Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis. Lab report

In the Hill reactions, chlorophyll molecules are oxidized by light, water is hydrolyzed, oxygen is produced, and the electrons and hydrogen ions are transported down a series of transmembrane electron carriers in the thylakoid membranes . The electrons terminate their travels by reducing NADP + to NADPH (a soluble electron carrier), which is then consumed by the Calvin Cycle in the stroma of the chloroplast. (See Figure 3 on Page 34 ) The electron transport proteins in the thylakoid membrane are asymmetrically arranged, and some of them can transport both hydrogen ions (protons) and electrons whereas some can transport only electrons. The arrangement of the thylakoid electron transport chain promotes the pumping of protons from the stroma into the thylakoid space . This translocation of hydrogen ions causes the lumen of the thylakoid sacs to be acidic (approximately pH 3.5) whereas the stroma is alkaline (approximately pH 8.0).

In 1961 Peter Mitchell and Jennifer Moyle proposed the chemiosmotic hypothesis . In essence, the hypothesis states that photosynthetic electron transport results in the accumulation of protons in the lumen of the thylakoids, and that this steep proton gradient is capable of generating ATP. They suggested that the proton motive force (PMF) drives protons through large thylakoid membrane complexes, called ATP synthases , from the lumen of the thylakoids back towards the stroma. For every three protons that pass through the ATP synthase, approximately one ATP is generated.

While protons are only translocated into the lumen of the chloroplast during the Hill reactions, protons will move through ATP synthase whenever there is a concentration gradient present, regardless of whether light is present or not. In this lab, we will isolate chloroplasts from spinach leaves and illuminate them with a strong light source in order to examine if the changes in pH that we observe are consistent with the chemiosmotic hypothesis . PROCEDURES i. Isolating chloroplasts from spinach leaves 1. At your bench, there will be 35 g of spinach leaves floating on the surface of a bowl of water. These leaves were left in the cold and dark overnight to reduce the starch content, and have been sitting for a couple of hours under a light source to activate the chloroplasts. Remove the leaves from the water.

2. Rip the leaves into small pieces, and then transfer the pieces to a chilled blender cup, and add 100 ml of homogenizing medium .

3. Blend at top speed for three 10 second bursts . Remove the blender cup, and pour the contents through three layers of cheesecloth into a chilled 250 ml beaker. Squeeze the cheesecloth gently to wring out as much fluid as you can.

4. Distribute the filtered homogenate equally into two 50 ml centrifuge tubes. Remember to balance your tubes by redistributing the homogenate as needed and using the triple-beam balance prior to each centrifugation!

5. Centrifuge at 500 x g for 2 minutes at 4 o C .

6. Transfer the supernatants to two clean 50 ml centrifuge tubes. Discard the pellets .

Centrifuge the supernatants at 2000 x g for 10 minutes at 4 o C to pellet the chloroplasts.

7. Discard the supernatants . Add 1.5 ml of 0.01 M NaCl to each tube. Resuspend the chloroplast pellets gently, by sucking them up and down into a Pasteur pipette. Then add a further 6 ml of 0.01 M NaCl to each tube. Place the tubes in an ice bath, and let them incubate for 10 minutes . This treatment swells the chloroplasts slightly, and makes the outer membrane leaky, so that any pH changes in the stroma can be detected in the external medium.

8. Centrifuge the chloroplast suspension at 10,000 x g for 5 minutes at 4 o C . Discard the supernatants . Resuspend the pellets by adding 2 ml of 0.01 M NaCl to each tube and sucking them up and down gently with a Pasteur pipette. Combine the re-suspended pellets into one tube, and store them on ice. This is your osmotically swollen chloroplast preparation.

ii. Chlorophyll determination Before doing the pH assay, it is necessary to ensure that the concentration of chlorophyll is 0.5 mg chlorophyll/ml . This will allow us to standardize our data across multiple trials.

1. Add 0.1 ml of the swollen chloroplast suspension to 19.9 ml of 80% acetone in a graduated cylinder. Cap the cylinder with Parafilm® and shake vigorously.

2. Pour a small volume of the acetone extract into a glass spectrophotometer cuvette ( acetone will dissolve plastic!!) and measure the absorbance of the solution at a wavelength of 654 nm . Use 80% acetone as a blank.

3. Take the absorbance value you have observed, and multiply it by 5.6 . The product gives you the concentration of chlorophyll in your suspension in mg/mL.

4. Dilute the chloroplast suspension ( NOT THE CHLOROPLASTS IN ACETONE ) as needed (use the formula C 1 V 1 = C 2 V 2 ) with enough cold 0.01 M NaCl to bring the final chlorophyll concentration to 0.5 mg/ml. You will need to make sufficient chloroplast suspension for a minimum of two trials, so make 15 mL in case you need extra .

5. Return the tube to the ice bath. Cover the diluted chloroplasts with aluminum foil until you are ready to do the pH assays.

2 iii. pH assays Important: Do not make the beakers for this section until you need them. (if they sit too long, they may not work) The solution should be a slight green colour, not blue.

1. The pH meter should already be turned on and in "standby" mode. The pH electrode will be sitting in a standard pH solution of pH 7.0. Please note that when removing the pH electrode from any solution, rinse it well with a squeeze bottle of distilled water and remove any residual water with a Kim Wipe before placing it into another solution. Refer to the pH meter meter operating instructions found in Appendix F .

2. To two 10 ml beakers, add the following:

1. A small ("flea") magnetic stir bar (be sure not to dump this down the sink after the assay) 2. 1.1 ml 0.048 M NaCl 3. 0.6 ml phenazine metosulphate (PMS) ( TOXIC!!-be careful ) 4. 3.6 ml distilled water 3. Place the beaker on a stir plate. Turn on the stir plate and get the stir bar moving at a moderate speed. Carefully lower the pH electrode into the solution. 4. Add 3.7 ml of your chloroplast suspension using the Gilson pipet provided. Turn on the light source , and position it as close as you can to the beaker.

5. Record the change in pH every 10 seconds for 2 minutes in total using the data table provided on the bench. Then turn off the light , and use the garbage bag provided to make a lightproof curtain over the box. Again, record the change in pH every 10 seconds for 2 minutes . Remove the curtain, and turn the light source back on . Continue to record the change in pH every 10 seconds for a final 2 minutes .

6. Do a second experiment by repeating steps 2-4 above with another beaker and a fresh preparation of chloroplasts. Using the second data table, record the pH at 10 second intervals for 2 minutes, with the light on . Add 20 microliters of carbonyl cyanide m- chlorophenyl hydrazone (CCCP) (a proton ionophore) using the Gilson micropipette provided. Continue to record the change in pH at 10 second intervals for another 2 minutes, with the light on .

Prior to the Lab 4B Tutorial please view the pH Assay video. You may wish to refer to Appendix F: Use of the pH Meter as well.

Lab 4 Report Labs 4A and 4B will require the submission of a formal lab report worth 10% of your final grade. The Lab 4 Report Requirements are available in the Lab 4 folder. You should review the General Lab Report Guidelines as well. Data analysis will be discussed during the Lab 4B Tutorial. 3