Laboratory 2 Chloroplasts and Photoreduction

Biology 29 Cell Structure and Function Spring, 2009 LABORATORY 2:CHLOROPLASTS AND PHOTOREDUCTION In this laboratory we will purify chloroplasts from spinach by differential centrifugation, then the chloroplast preparation will be used to demonstrate the generation of reducing power by chloroplasts in the light. Part I: Purification of chloroplasts General comments In order to retain complex biochemical activities in an extract, it is important to prevent disruption or degradation of the structural integrity of the fractions. In this procedure, we avoid disruption by (1) the use of a buffer (Buffer A) wtih a moderately high salt concentration (0.35 M NaCl) to protect the chloroplasts from osmotic shock, and (2) depending on you to not subject your samples to extreme mechanical stress. In particular, do not over-homogenize the leaf material, doing so can actually reduce your yield – the blender procedure below will yield plenty of chloroplasts for your needs. The second problem, degradation, is a much more pervasive one than the first, with a simple cure -- ice. Everyone is provided with an ice bucket and access to a refrigerator. At every step, it is profitable to make use of your ice: prechill tubes, beakers, and flasks; keep all the intermediate materials on ice while waiting for the next step; use the ice bucket as your test tube rack. In addition, the principal source of contamination is fingers -- when you need to stick something into your prep, use a pasteur pipette. Step 1: Crude homogenate Weigh out about one 250 ml beaker full of spinach leaf tissue (roughly 25 grams). This tissue should be torn from healthy looking leaves, avoiding stems and large veins. After weighing, rinse the tissue well with distilled water. From this point on, hands off. Transfer the leaf tissue to the Waring blender container. Add 100 ml of chilled Buffer A. Blend at low to moderate speed until all of the tissue has been dragged into the general mixture (about 10 seconds). Then blend at high speed for 30 seconds. Transfer this homogenate to a chilled beaker. Rinse the Waring jar, first with tap water, then distilled. Leave it on ice for the next group who is ready for it. Remove an aliquot of this homogenate for later inspection in the microscope. Filter the rest through a piece of Mira cloth into a second beaker in the ice bucket. This filtered material is the final crude homogenate. Remove an aliquot of it as well. 1 Step 2: Removal of nuclei, unbroken cells, and debris Decant the crude homogenate into two centrifuge tubes and balance the tubes by eye. Take them to the centrifuge, where they will be spun at 1250 RPM for two minutes, and allowed to coast to a stop without braking. Keep track of the outside of the tubes, since the pellets will be on this side. In one smooth motion, decant the supernatants away from the pellets into two fresh centrifuge tubes. Remove aliquots of supernatant and pellet for microscopic analysis. Step 3: Chloroplasts The supernatants from the last step should be taken to the centrifuge for spinning at 3500 RPM for five minutes. At this speed, most of the chloroplasts should be pelleted. Using the same technique as in 2), decant away the supernatants into a (cleaned) beaker. To resuspend the chloroplast pellets, make a "brush" by inserting a small piece of cotton into the blunt end of a Pasteur pipette, and wet this brush with a little Buffer A. Add 1 ml of Buffer A to each of the two centrifuge tubes and gently brush the pellets into this buffer until homogeneous. Combine the resuspended chloroplasts into one small tube. Again, save appropriate aliquots. Step 4: Microscopic analysis Examine all of the aliquots taken under phase contrast. From this inspection, try to estimate the purity of the isolated chloroplasts, and the relative efficacy and yield of the various purification steps. 2 PART II: Electron transport General comments In the normal chloroplast, the reducing power generated by light is used to reduce CO2 to carbohydrate, a process not easily monitored in an afternoon laboratory. Instead, we will open up the chloroplasts to admit a dye, dichlorophenolindophenol (DCPIP) which the chloroplasts can reduce. This dye is blue; its reduced form is colorless. The transition can be easily monitored by measuring the absorbance at 600 nm. The general assay used here is as follows. About 10 µl of chloroplast suspension is mixed in a cuvette with 90 µl of water. The osmotic shock of this dilution breaks the chloroplasts. This suspension is then diluted to 3 ml with a phosphate buffer (Buffer B), and about 0.15 ml of 1 mM DCPIP is added. After prompt mixing by inversion of the cuvette over parafilm, the cuvette is irradiated with light, and at intervals (5 seconds - 10 minutes) inserted into the spectrophotometer and the absorbance at 600 nm measured. The proportions of chloroplasts and DCPIP should be varied in preliminary experiments to yield optimal conditions (reaction completion in about 5-10 minutes, starting absorbance between 0.5 and 1). The blank chosen for all experiments is critical. Decide on an appropriate blank to be used and then check with an instructor or assistant. Section 1: Light dependence Compare the kinetics of dye reduction in the light and the dark. Design and carry out an experiment to test the dependence of the rate of reduction on the light intensity. Determine whether the rate is dependent on the amount of chloroplast suspension used. Section 2: Uncoupler The presence of NH3 in the medium "uncouples" the synthesis of ATP from the transport of electrons. Using 0.1 mM (NH4)2SO4 and high light intensity, measure the rate of electron transport and compare it with the normal rate. Interesting additional experiments might include comparing the rates of coupled and uncoupled transport at low light intensities and in the dark. Section 3: Oxygen evolution There are certain compounds (herbicides) which directly block the reaction which is responsible for the evolution of O2 by chloroplasts. One is 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). Measure the rate of dye reduction in the presence of 0.03 mM DCMU. Interesting additional experiments might include measurement of the dependence on light intensity and DCMU concentration. 3 4 Laboratory 3 (Chloroplast reduction) Assignment (30 pts total) Due date - for Kinesins: March 12; for Dyneins: March 26 The assignment for this laboratory includes the following: For each section (Section I: Light Dependence, Section II: Uncoupler, Section III: Oxygen Evolution), choose a graph that summarizes one of your findings, and write a brief conclusion (one paragraph) of your findings Graphs: Otherwise known as Figures, may be prepared on the computer (Excel, etc) or otherwise neatly hand-done. Remember to properly label the axes of the graphs and to include proper units. Each figure should have a legend: beginning with (1) a figure number, followed by (2) a descriptive title (an incomplete sentence) specific to your data, and then (3) the rest of the legend in complete sentences. The legend is found at the bottom of the figure. Figure legends should succinctly explain the experiment, as well as how results were collected, calculated, and presented. Optional: Appendix: Your figure and conclusion should be sufficient to illustrate your results (if not, you may not have summarized your findings sufficiently). However, if you wish to provide additional information (ables, sample calculations, notes about purification), use an appendix but please keep it brief. Tables: Are not necessary for this report. However, If you wish to provide data in tables, these may be typed (Excel, Word, etc.) or neatly hand-written. Remember to include appropriate units for the values in your table. Each table should be numbered (Table 1, Table 2, etc.) and have an informative (specific) title at the top. Tables do not have a legend; however, any symbols used should be defined in footnotes at the bottom of the table. 5