Measuring Photosynthetic Electron Transport (Hill reaction)

In the 1937, Robert Hill, an English biochemist, made two milestone discoveries about the photosynthetic reactions. First, Hill and his coworkers showed that O2 released during photosynthesis was actually derived from H2O, not CO2. They also demonstrated that the light absorbing process can be separated from the reactions in which CO2 is "fixed" into carbohydrates. The Light-Dependent Reactions In the first stage, called the light-dependent reactions, light is absorbed by the pigment molecules (such as chlorophyll), and the energy is transferred to molecules of ATP and NADPH: 2 H2O + 3 ADP + NADP + 3 Pi
light

> O2 + 3 ATP + NADPH2

During the these reactions, energy from light is absorbed by pigment molecules and transferred to electrons of a water molecule. This energization causes the water molecules to split, releasing O and H, and the energy carried in the electrons is ultimately used to produce ATP and NADPH2. The oxygen given off during photosynthesis is merely a by-product. The "splitting" of water (photolysis) into oxygen and hydrogen is illustrated in Figure 1. Figure 1. Photolysis of water during the light reactions.
SUNLIGHT

O2 e- (electrons) ATP and NADPH

2H2O 4H+ The Carbon Fixation Pathway

The ATP and NADPH produced during the light-dependent reactions are used during the second stage of photosynthesis, called carbon fixation, to convert CO2 into carbohydrates: 3 CO2 + 9 ATP + 6 NADPH2 + 3H2O
light

> 3-PGA + 9 ADP + 6 NADP + 8 P

The light reactions of photosynthesis occur on the Thylakoid membranes in the center of the chloroplast, and carbon fixation occurs in the surrounding region, called the stroma (Figure 2). Measuring Ps Electron Transport page 1

The Hill Reaction Robert Hill showed that the two stages of photosynthesis could be "uncoupled" if the thylakoid membranes and the stroma components of chloroplasts are separated via cell fractionation. Using isolated thylakoids, the light reaction will proceed without carbon fixation in the presence of light. This type of in vitro reaction is called the Hill reaction. The Hill reaction will occur if isolated chloroplasts are provided with a chemical, called a Hill reagent, that accepts electrons from the photosynthetic electron transport pathway. The Hill reagent that we will use is DCPIP (2,6dichlorophenol-indolphenol). DCPIP is blue in its oxidized form, and becomes colorless when it is reduced during the Hill reaction.

Figure 2. Simplified drawing of the structure of a chloroplast.

DCPIP(oxidized) + 2 e- > DCPIP(reduced) Blue Colorless Thus, the rate at which electron transport occurs in the Hill reaction can be measured spectrophotometrically (at 620 nm) by following the change in absorbance of DCPIP as it accepts electrons from the electron transport chain. To perform the Hill reaction, a sample of a chloroplast suspension will be mixed with the Hill reaction buffer (containing DCPIP) and exposed to light for a series of 30 second intervals. After each exposure period, the absorbance of the DCPIP will be measured. The absorbance values can then be plotted versus time to determine the rate of DCPIP reduction as a measure of PET (Figure 3).

1 Abs (620 nm) 0.8 0.6 0.4 0.2 0 0 2 4 6 8 Time (min.) 10 12 y = -0.0761x + 0.968

Figure 3. Hill reaction data plotted with trend line through linear portion. The absolute value of the slope (0.0761) is the rate of change in DCPIP absorbance.

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Determining the rate of electron transport The rate of the electron transport can be calculated from the slope of the line (Figure 3), which has units of ABS DCPIP / min. However, the rate observed is also a function of the light intensity and the amount of chloroplasts present. The amount of light striking the sample can measured with a light meter as the photon fluence rate (M m-2s-1). The amount of chlorophyll in the sample, which can be measure spectrophotometrically, will be proportional to the amount of chloroplasts. When these values are factored in, a normalized rate can be calculated with units of ABS DCPIP / min / mg chlorophyll / M m-2s-1. Objectives 1. To learn about cell fractionation and how isolated thylakoid membranes that 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 examine the effects of certain plant herbicides on photosynthetic electron transport.

Overview of the Lab Exercise The steps of the experiment are as follows: I. Prepare a chloroplast fraction through cell fractionation II. Determine the chlorophyll concentration of your sample III. Measure rate of Hill reaction 1. Set up the light source 2. Zero the spectrophotometer with a fully reduced assay sample 3. Make repeated measurements of Hill reaction in the presence and absence of light, to determine the rate of PET and to demonstrate the light dependence of the Hill reaction. IV. Determine which herbicides inhibit the light-dependent reactions of photosynthesis

Procedures
I. Preparing the Chloroplasts
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 prechilled.

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Supplies
homogenization buffer (which you prepared) 2 polypropylene centrifuge tubes foil 50 ml graduated cylinder plastic funnel 15 ml Potter-Elvehjem homogenizer misc pipets ice bucket 1 13mm test tube double layer of cheese cloth 100 ml beaker micropipetters

1. Homogenize the spinach leaves (this is done in batch for the whole class) 1. Fresh spinach leaves are washed with roH2O 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. 3. The leaves will be homogenized with the blender set at its highest speed for 10 second periods, ~four times, interrupted with brief pauses.

2. Fractionate the homogenate 4. Each group will filter enough homogenate through a double layer of cheesecloth into a 50 ml graduated cylinder (embedded in ice) to yield 40 ml. 5. Transfer the filtered homogenate to a polypropylene centrifuge tube. 6. Counterbalance your tube against that of another group and put the tubes in opposite positions in the rotor (SU-34). Centrifuge at 500 x g (2000 rpm) for 1 minute. 7. Carefully decant the supernatant into a clean centrifuge tube. 8. Discard the pellet in the sink. This pellet contains whole cells, starch grains, and nuclei. 9. Centrifuge the low-speed supernatant 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.

3. Resuspend the chloroplasts in an isotonic solution 1. Carefully pour off the supernatant from the centrifuge tube. 2. 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. 3. Transfer the chloroplast suspension to a Potter-Elvehjem homogenizer. 4. Rinse the centrifuge tube with an additional 2 ml of Homogenization Buffer, and then combine this with the rest in the homogenizer 5. Fully resuspend the chloroplasts with 3 - 4 passes of the pestle. 6. Using a Pasteur pipet, transfer the chloroplast suspension into a 13 mm test tube wrapped in foil and place it in your ice bucket.

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II. Determine the Chlorophyll Concentration of the Chloroplast Suspension When performing the Hill reaction, two factors in particular will influence the rate of photosynthetic electron transport (i.e., DCPIP reduction) that is observed the amount of chloroplasts added to the reaction mixture and the intensity (irradiance) of the light. The concentration of chlorophyll in the suspension is a reliable measure of the amount of chloroplasts., so this need to be determined. Later, you will divide the rate of DCPIP reduction by the amount chlorophyll you add to the reaction mixture, to express the rate of the Hill reaction per mg chlorophyll. Supplies 2 cuvettes 1 screwcap test tubes P-200 pipetman 80% acetone table-top centrifuge spectrophotometer 10 ml pipet

1. With a pipet, using a P-200 pipetter, transfer 100 L of your suspension to a screw cap test 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 the space provided on calculations page at the end of the exercise, and perform the necessary calculations after the lab exercise is completed. Note: The concentration of the chlorophyll has been dramatically diluted by the acetone, and the dilution factor must used in later calculations. The dilution factor can be calculated by dividing the final volume of the diluent by the original volume of the sample diluted (being sure that units agree).

Hill Assay Buffer Component Concentration Tricine 50 mM NH4Cl 1 mM Sucrose 100 mM DCPIP ~100 M

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III. Measuring the Hill Reaction

Supplies
5 ml pipet P-20 pipetter water bath heat sink cuvette holder Na Dithionite (Na Hydrosulfite) solution (Pink cap) Hill reaction buffer light source

1. Set up the light source

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.

2. 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 cuvette. 3. Add 15 l 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 experiment (but not between individual measurements).

3. Measuring the Hill Reaction

Each time you measure the Hill reaction, you will do the following things. 1. Zero the spectrophotometer at 620 nm against the Hill Reaction blank (prepared above). 2. Add 3 ml of Hill Reaction Buffer to a cuvette. 3. Add 15 L of your chloroplast suspension (do not add 80% acetone solution!); cover the cuvette with parafilm and invert twice to mix the solution. 4. Measure and record (in Raw Data Table at end of exercise) the absorbance of the complete reaction mixture before exposing it to the light. The absorbance should be around 1.0. 5. Place the tube at a spot 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 multiple times.

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4. During the first Hill Reaction assays, adjust the rate of PET
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 of about 0.05 unit per 30 seconds as in Figure 3. If the rate of the Hill reaction is too fast or too slow, adjust the light level on the halogen lamp. Performing this assay should take no longer than 15- 20 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 about 0.15 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.

5. Collect a complete data set for calculating the Hill reaction rate
After you have established the proper conditions to yield a acceptable rate of DCPIP, repeat the experiment to obtain a complete set of data (from the initial absorbance to about 0.2) for determination of the Hill reaction rate.

6. Determine if the results are light-dependent

Repeat the experiment, but this time with a layer of foil in front of the cuvette. Collect data for about 4 minutes.

7. Measure the light level

The appropriate light parameter is photon fluence rate (micromoles of photons per square-meter per second, i.e., M m-2s-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/m2), conversion to a fluence rate is accomplished by dividing the measurement by 50.

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8. Calculate the rate of the Hill reaction

These Calculations Can Be Performed after Completing the Exercise. They should be done on the provided calculations page. As explained above, the Hill reaction rate will have in units of " ABS DCPIP / min/ g chlorophyll / M m-2s-1" Thus, you will need to determine: 1. The rate of DCPIP reduction ( ABS / min). This will be determined from the slope of the trendline drawn through the linear part of the data set. (The slope, of course, is m in the equation y=mx+b.) Be sure to use the absolute value of the slope as calculated, it will be negative because the absorbance of DCPIP decreases during the reaction 2. Total mg of chlorophyll used in the Hill reaction mixture. This is calculated from the chlorophyll measurement using the absorption coefficient. 3. The light level in units of photon fluence rate (which has units of M m-2s-1) , calculated from the light measurement

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IV. THE EFFECT OF HERBICIDES ON THE HILL REACTION

A number of common agricultural herbicides owe their toxicity to inhibition of photosynthetic electron transport (PET). By studying their effects upon the Hill reaction, it should therefore be possible to determine which herbicides owe their toxicity to blockage of PET. The herbicides we will study are: 2,4-D: A widely used herbicide. When it was combined with 2,4,5-T (a much more toxic substance) the mixture was known as "Agent Orange" a powerful defoliant, as Vietnam discovered in the 1960s. Atrazine: Atrazine is the active ingredient of the herbicide called "Roundup". Caution: Atrazine is a suspected carcinogen

To test the effect of the herbicides on the Hill reaction, you will start the assay in the absence of the herbicide, and collect data until the absorbance decreases to 0.5 unit. At this point the herbicide will be added, and the rate of the Hill reaction will be further examined. Idealized effects of two herbicides are shown in Figure 4. Notice that addition of herbicide XXX had no effect on the Hill reaction. However herbicide ZZZ caused the Hill reaction to stop, and, therefore, would be interpreted to be a PET inhibitor. Sometimes a slight reoxidation of DCPIP will occur, which will cause the absorbance to slowly increase after the inhibitor has been added.

Figure 4. Possible effects of herbicides on the Hill reaction.

1
(non-PET inhibitor) (PET inhibitor)

Abs (620 nm)

0.8 0.6
ZZZ

0.4 0.2 0 0

XXX

10 Time (min.)

15

20

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Supplies
P-20 or P-200 Pipetman herbicide solutions (front bench) Pipetman P-20 chloroplast sample cuvettes methanol control

Procedure
Examine the effect of each herbicide separately. Follow the following steps for each tube. 1. Prepare an assay mixture as described above. 2. Measure and record the absorbance at appropriate time intervals until the absorbance decreases to 0.5 Abs unit. 3. Using a Pipetman, add 15 l of the appropriate herbicide. Cover the tube and invert twice to mix the sample. 4. Continue to measure the Hill reaction until the absorbance reaches 0.2 unit, or the Hill reaction can be seen to have stopped. 5. Repeat steps 1 - 4 for each herbicide and the methanol control. Be sure to use a new pipet tip for each herbicide. 6. Plot the data and determine if each herbicide is a PET inhibitor.

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TABLES FOR RAW DATA

Sample: _________ TIME ABS Sample: _________ TIME ABS Sample: _________ TIME ABS Sample: _________ TIME ABS

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Sample: _________ TIME ABS

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Sample: _________ TIME ABS

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Sample: _________ TIME ABS

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Sample: _________ TIME ABS

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Names: ___________________________ A. Using the Beer-Lambert law, calculate the chlorophyll concentration of your chloroplast suspension. The absorption coefficient for chlorophyll at 652 nm is 35.4 ml cm-1 mg-1. ABS652 = _______ dilution factor =_______

concentration of chlorophyll = ________ mg/ml (show calculations below) (Concentration should be between 0 and 10 mg/ml.)

B. Use above value to calculate the mg of chlorophyll in the chloroplast suspension added to each Hill assay mixture: ( _____ mg/ml Chl ) X ( ____ ml of chloroplast added to assay) = ________ mg chlorophyll in assay C. Convert to light measurement to photon fluence rate: Light intensity: ________ lux To convert into photon fluence rate (M photons m-2 s), divide lux by 50: Photon Fluence Rate = _____ lux 50 = ______ M m-2 s-1 D. Determine the rate of DCPIP reduction From your graph, obtain the slope of a trendline drawn through the linear part of the data (see Figure 3). Use the absolute value of the slope. Why? Slope = ________ ABS DCPIP / min ***NOTE: Reaction rate is calculated per minute, not second.*** E. Complete the calculation of rate by factoring in chlorophyll and irradiance A. Divide the DCPIP reduction rate by the mg of chlorophyll in the assay: ___________ ABS DCPIP / min / mg Chlorophyll B. Now divide this number by the photon fluence rate to calculate Hill reaction rate: ___________ ABS DCPIP / min / mg Chlorophyll / M m-2s-1 (correct calculations should yield a number between 0 and 1)

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What to turn in: (20 points)

A full lab report is not required. Students can work together on calculations, but must prepare graphs independently. 1. All the values and calculations (A - E, pg 12) leading to the final Hill reaction rate, neatly prepared. 2. A graph showing the Hill reaction in the presence and absence of light (from Part III). Draw trendline only through the linear part of the graph, display equation on the graph. Use the absolute value of the slope (remember y = mx + b) to calculate the rate of the Hill Reaction. Be sure to format graph properly and include a Figure legend. 3. A graph showing the effects of atrazine, 2,4-D and methanol on the Hill reaction (from Part IV). Plot all three sets of data together in the same graph, offset as shown in Figure 4. This can be accomplished by arbitrarily assigning staggered time frames for each data set. Mark with an arrow when the inhibitors (or methanol) were added. Indicate on the graph whether the herbicide is a PET inhibitor or not. Be sure to format graphs properly and include a figure legend.

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