Studying Photosynthetic Electron Transport through the Hill Reaction

Transcription

1 Studying Photosynthetic Electron Transport through the Hill Reaction In this lab exercise you will learn techniques for studying electron transport. While our research system will be chloroplasts and photosynthetic electron transport (PET), many of the basic research principles apply to the study of mitochondrial electron transport. The Hill Reaction If thylakoids are separated from the stroma of chloroplasts, the light-dependent reactions of photosynthesis could be studied independent from the CO 2 fixation reactions. As you will recall, in the light-dependent reactions, electrons extracted from water in the PSII reaction center pass through an electron transport pathway and eventually to NADPH. (Figure 1). Electron transport also produces an hydrogen ion gradient used for ATP synthesis. Given, light and a supply of NADP, isolated thylakoids will carry out photosynthetic electron transport (PET), and reduce NADP to NADPH. But measuring NADP reduction is difficult. Figure 1. Z-scheme presentation of photosynthetic electron transport. In the 1930s Robert Hill showed that if isolated thylakoids are combined with dye that changes colors when it accepts electrons, electron flow could be measured directly (and more simply) with a spectrophotometer. The molecule that he used is DCPIP (2,6-dichlorophenol-indolphenol). When DCPIP accepts electrons, it changes from a blue to a colorless state DCPIP (oxidized) + 2 e - > DCPIP (reduced) Blue Colorless and the change can be monitored spectrophotometrically at 620 nm. The In vitro photosynthetic electron transport process from water to DCPIP is called the Hill reaction. Abs DCPIP (620 nm) y = x Time (min) Figure 2. Change in absorbance as DCPIP is reduced during the Hill reaction. To perform the Hill reaction, thylakoids are combined with the Hill reaction assay buffer (containing DCPIP) and exposed to light. At 30 second intervals the absorbance of the DCPIP is measured. When the absorbance values are plotted versus time (Figure 2), the slope of the line is a measure of the rate of PET from water to DCPIP. Measuring the Hill Reaction Page 1

2 Actually, we will not prepare a suspension of pure thylakoids. Purified thylakoids are very difficult to prepare, and not actually needed. Instead, we will prepare a suspension of chloroplasts treated to expose the thylakoids. Hill assay buffer is slightly hypotonic and causes the chloroplast outer membranes to rupture, releasing the stromal enzymes and allowing the thylakoids to be exposed to the Hill reaction buffer (including the DCPIP). The amount of thylakoids added to the assay mixture and the intensity of the light will affect the observed rate of the Hill reaction, thus these must be measured and factored into the calculation of the Hill reaction rate. The amount of thylakoids is typically determined by measuring the concentration of chlorophyll in the suspension, and light intensity will be measured with a photometer. The final Hill reaction rate will have the units: Δ ABS DCPIP / min / mg chlorophyll / μm m -2 s -1 The Hill reaction as an analytical tool The Hill reaction can be used to study electron transport, such as how different physical or biochemical factors influence the rate of electron transport. However, interpretations must be made cautiously since the Hill reaction is not the same as normal PET. DCPIP does not accept electrons at the end of the chain (like NADP), but rather somewhere in the middle, and thus the Hill reaction does not involve all components of the PET chain. And this begs the question, which components of the PET chain participate in the Hill reaction, and how can this be determined? One way to dissect PET is with PET inhibitors chemicals that block electron transport at specific locations. Because of their effect on PET, some of these chemicals are used commercially as herbicides. In the presence of chemical that inhibits PET prior to the DCPIP accepting site, DCPIP reduction will be blocked. Consider these three inhibitors and where they block PET (Figure 3): DCMU an herbicide that blocks electron flow to plastoquinone. DBMIB blocks electron flow to cytochrome b 6 -f. Bipyridylium herbicides (which includes the herbicide paraquat) block electron flow to ferrodoxin. Figure 3. Blockage sites in PET of three inhibitors. Hypothesis: We will hypothesize that DCPIP accepts electrons from cytochrome b 6 -f,. References Dayan FE, Duke SO, Grossmann K Herbicides as Probes in Plant Biology. Weed Science 58: Trebst A Inhibitors in the functional dissection of the photosynthetic electron transport system. Photosynthesis Research 92(2): Measuring the Hill Reaction Page 2

3 Objectives 1. To learn about cell fractionation and how thylakoids can be used for investigation of the photosynthetic reactions. 2. To learn how quantitative measurements of photosynthetic electron transport can be made through the Hill reaction. 3. To learn how electron transport inhibitors can be used to dissect photosynthetic electron transport. The steps of this lab are: 1. Preparation of chloroplasts 2. Hill reaction assay setup 3. Measurements of chlorophyll concentration and light intensity 4. Hill reaction assay measurements 5. Hill reaction assay without light and with inhibitors Using the Genesys 20 Spectrophotometer 1. Turn on the spectrophotometer. Allow to warm up about 15 minutes (stabilizes light source). 2. Set to proper wavelength. 3. Transfer the blank (control) sample to a cuvette, place it in the sample holder, and set the absorbance to Replace the sample with the test sample and record the absorbance. Supplies Homogenization buffer (which you prepared) polypropylene centrifuge tube 15 ml Potter-Elvehjem homogenizer 50 ml graduated cylinder Foil ice bucket 100 ml beaker plastic funnel 13mm test tube misc pipets Procedures Measuring the Hill Reaction Page 3

4 1. Preparing the chloroplast suspension In the first part of the exercise, spinach leaf cells will be fractionated to yield a chloroplast fraction stabilized in an isotonic solution. The homogenate and chloroplast suspension should be kept cold (in an ice bucket) at all times. Homogenization buffer should be pre-chilled. Homogenize the spinach leaves (this is done in batch for the whole class) 1. Fresh spinach leaves are washed with roh 2 O and then stored in the refrigerator overnight. 2. In a blender, 100 g of de-veined leaves will be combined with 300 ml of homogenization buffer. (Homogenization buffer is isotonic to the cell cytosol, preventing rupturing of the chloroplasts during preparation.) 3. The leaves will be homogenized with the blender set at its highest speed for 10 second periods, ~four times, interrupted with brief pauses. 4. The homogenate is then passed through a cloth filter to remove larger particulate materials. Each group will receive 40 ml of the filtered homogenate. Centrifuge the filtered homogenate and resuspend the chloroplasts 5. Transfer the filtered homogenate to a polypropylene centrifuge tube. 6. Counterbalance your tube against that of another group and put the tubes opposite each other in the centrifuge SS-34 rotor. 7. Centrifuge the filtrate at 2,000 x g (4,250 rpm) for 10 minutes. In this step the chloroplasts in the homogenate sediment to the bottom of the tube. 8. Carefully pour off the supernatant from the centrifuge tube. 9. Add 2.0 ml of homogenization Buffer to the pellet, and resuspend the chloroplast pellet by gently sucking it into and out of a Pasteur pipet. 10. Transfer the chloroplast suspension to a Potter-Elvehjem homogenizer. 11. Rinse the centrifuge tube with an additional 2 ml of Homogenization Buffer, and then combine this with the rest in the homogenizer 12. Fully resuspend the chloroplasts with 3-4 passes of the pestle. 13. Using a Pasteur pipet, transfer the chloroplast suspension into a 13 mm test tube wrapped in foil and place it in your ice bucket. 2. Determine the chlorophyll concentration of the chloroplast suspension As described in the introduction, the relative amount of thylakoids in your sample can be measured at the concentration of chlorophyll in the suspension. Chlorophyll can be measured by dissolving a volume of your sample in 80% acetone and measuring the absorbance at 652 nm. Using the Beer-Lambert law and an absorption coefficient of 35.4 ml cm -1 mg -1, the concentration of chlorophyll in the acetone can be calculated. Supplies 2 cuvettes 80% acetone spectrophotometer 1 screwcap test tubes table-top centrifuge 10 ml pipet P-200 pipetman glass cuvettes Measuring chlorophyll concentration 1. With a pipet, using a P-200 pipetter, transfer 100 μl of your suspension to a screw cap test Measuring the Hill Reaction Page 4

5 tube containing 9.9 ml of 80% acetone. Do not chill this solution. 2. Place the tube in the table-top centrifuge opposite that of another group and centrifuge at 2000 rpm for 5 minutes. 3. Using a blank, zero the spectrophotometer at a wavelength of 652 nm, and then measure the absorbance of the chlorophyll extract. 4. Record the absorbance and the dilution factor in your lab notebook, and perform the necessary calculations after the lab exercise is completed. 3. Setting up for and measuring the Hill reaction rate Supplies 5 ml pipet Hill reaction buffer P-20 pipetter light source water bath heat sink 2 plastic cuvettes cuvette holder Na Dithionite (Na Hydrosulfite) solution (Pink cap) Hill Assay Buffer Component Concentration Tricine 50 mm NH 4 Cl 1 mm Sucrose 100 mm DCPIP 80 μm Set up the light source as illustrated in class. 1. The cuvette holder should be about 2 feet from the light. Tape it to the bench top. 2. Place a heat sink water bath about 6" in front of the cuvette. 3. Make sure the light beam shines directly on the cuvette. Create a blank and zero the spectrophotometer The spectrophotometer must be zeroed against the assay mixture containing fully reduced (clear) DCPIP. The simplest way to fully (and quickly) reduce the DCPIP is to add a strong reducing agent, such as sodium dithionite. 1. Set the wavelength of the spectrophotometer at 620 nm. 2. Place 3.0 ml of Hill reaction buffer to a clean plastic cuvette. 3. Add 15 μl of chloroplast suspension (not the acetone solution) and 1-2 drops of dithionite solution. Mix until blue color disappears. 4. Zero the spectrophotometer using this tube. Save the blank. The spectrophotometer should be re-zeroed before each Hill reaction assay (but not between individual measurements). Begin taking measurements of the Hill reaction. Measuring the Hill Reaction Page 5

6 The Hill reaction is measured by taking a series of absorbance measurements of a sample between exposures to light (the slope of the line from the measurements yields the rate of the Hill reaction). For each assay you will do the following things. Read through these steps before beginning. 1. Zero the spectrophotometer at 620 nm against the Hill Reaction blank. 2. Add 3 ml of Hill Reaction Buffer to a plastic cuvette. 3. Add 15 μl of your chloroplast suspension (do not add 80% acetone solution!); cover the cuvette with parafilm and invert twice to mix the solution. 4. Record the absorbance of the complete reaction mixture before exposing it to the light. The absorbance should be around Place the cuvette in the holder in the light path. 6. Time the exposure for 30 seconds. 7. Remeasure the absorbance, and record the time and absorbance. 8. Repeat steps 5 7 for 4 minutes (or a shorter period, during the initial set up of the light conditions as described in #1 below). 9. Discard assay mixture and reuse the cuvette. 1. Adjust the setup to obtain a suitable rate of DCPIP reduction Before a complete data-set can be collected for calculation of the rate of PET, the light intensity may need to be adjusted to get a reasonable rate of DCPIP reduction. We want a rate that yields a decrease in absorbance between Abs unit per 30 seconds as in Figure 4. If the rate of the Hill reaction is too fast or too slow, adjust the light level on the halogen lamp. Making these adjustments should take no longer than minutes. 1. Prepare a Hill Reaction assay mixture as described above 2. Measure the absorbance for three 30 second intervals. 3. If the absorbance has not dropped Abs unit between the initial and the final measurements, then increase or decrease the light intensity. Repeat steps 2 and 3, until a suitable rate of DCPIP reduction is achieved. 2. Collect two complete data sets for calculating the Hill reaction rate 1. After you have established the conditions yielding an acceptable rate of DCPIP reduction, repeat the experiment to obtain a complete set of data sets (4 minutes) for calculating the Hill reaction rate. 2. Repeat the assay with a fresh mixture. 3. After class graph the data and determine the slopes of the lines. 4. Measure the light level 1. The instructor will help you to measure the light intensity using a photometer. The appropriate light parameter is photon fluence rate (micromoles of photons per square- Measuring the Hill Reaction Page 6

7 meter per second ( i.e., μm m -2 s -1 ), the flux of photons at the point of measurement. Since the meter we have available yields units of light intensity (lux, or foot candles/m 2 ), conversion to a fluence rate is accomplished by dividing the measurement by Running the Control and studying the effect of the inhibitors Control: Determining if the DCPIP reduction is light-dependent 1. Repeat the Hill reaction assay, but this time with a layer of foil in front of the cuvette. Collect data for 3 minutes. Testing PET inhibitors In order to test the effects of the inhibitors, repeat the Hill reaction assay three times. For each inhibitor: Use a different cuvette for each assay. Why? The herbicides that will be tested are: DCMU, DBMIB, bipyridylium 1. Take measurements without the inhibitor for two minutes. 2. Add the 10μL of an inhibitor, mix, and take measurements for two additional minutes. 6. Calculating the Hill reaction rate The Beer-Lambert Equation The absorption coefficient (a) is property of a particular substance at a specific wavelength and under a specific set of conditions, such as ph, solvent, etc. The relationship between a, concentration (c), and absorbance is known as the Beer-Lambert Law: Abs = (a) x (b) x (c) where b equals the path length (the distance the light traverses through the sample), which is usually 1 centimeter for most cuvettes. You may have seen the equation written as Abs = bc; where is the molar extinction coefficient. We use a here because the concentration units of proteins are expressed as μg/μl (or mg/ml), not in molarity. When using the absorption coefficient to determine concentration, the equation can be rearranged to solve for c c = Abs ) (a x b), since b = 1, c = Abs ) a A. Using the Beer-Lambert law (Abs=concentration x absorption coefficient), calculate the chlorophyll concentration of your chloroplast suspension. The absorption coefficient for chlorophyll at 652 nm is 35.4 ml cm -1 mg -1. B. Use above value to calculate the mg of chlorophyll in the chloroplast suspension added to each Hill assay mixture: mg chl in assay = mg/ml Chl X ml of chloroplast sample added to assay C. Convert to light measurement to photon fluence rate: photon fluence rate (μm photons m -2 s -1 ) = divide lux by 50 D. Determine the rate of DCPIP reduction Your graph must have Abs graphed vs minute (not seconds). From your graph, obtain the slope of Measuring the Hill Reaction Page 7

8 a trendline (The slope, of course, is m in the equation y=mx+b), which has units of Δ ABS DCPIP / min. E. Complete the calculation of rate by factoring in chlorophyll and irradiance The Hill reaction rate has units of Δ ABS DCPIP / min / mg Chlorophyll / μm m -2 s -1 and is calculated as Slope / mg of chlorophyll / photon fluence rate. 1 Graphing multiple Hill reaction data sets. Often to conserve space, and when the specific x-axis values are not important, multiple data sets are graphed side-by side, as shown in Figure 4. This can be done by altering the start times, so as to offset the data. For inhibitor assays, trendlines are not included. Abs DCPIP (at 620 nm) Inhibitor 1 inhibitor Time (min) Figure 4. Effects of different PET inhibitors on the Hill reaction. The Hill reaction was allowed to proceed normally, and at the 1.5 minute time point 10μL of the indicated inhibitor was added. Measuring the Hill Reaction Page 8