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 CO2 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 Figure 1. Z-scheme presentation of photosynthetic electron
difficult.
transport.
1
Abs DCPIP (620 nm)
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.
0.9
y = -0.1309x + 1.0877
0.8
0.7
0.6
0.5
0
1
2
3
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
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-2s-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 b6-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 b6-f,.
References
Dayan FE, Duke SO, Grossmann K. 2010. Herbicides as Probes in Plant Biology. Weed Science 58:340–350.
Trebst A. 2007. Inhibitors in the functional dissection of the photosynthetic electron transport system. Photosynthesis
Research 92(2): 217-224.
Measuring the Hill Reaction
Page 2
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 0.
4. 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
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 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. (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
1 screwcap test tubes
P-200 pipetman
80% acetone
table-top centrifuge
glass cuvettes
spectrophotometer
10 ml pipet
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
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
1 mM
NH4Cl
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
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 1.0.
5. 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 0.05 – 0.1 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 10 - 15 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 0.05 – 0.1 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 squareMeasuring the Hill Reaction
Page 6
meter per second ( i.e., μM 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.
5. 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-2s-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
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-2s-1 ” and is
calculated as Slope / mg of chlorophyll / photon fluence rate.
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)
1
Inhibitor 1
0.9
inhibitor 2
0.8
0.7
0.6
0.5
0
1
2
3
4
5
6
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