3/26/06
Lab 5. Photosystem II
Lab 5.
Use of an Artificial Electron Acceptor to Study Photosystem II
READING: Campbell, N. A., Biology, 6th ed., Chapter 10
I.
BACKGROUND
The Reaction Center of Photosystem II
The most widely known attribute of green plant photosynthesis is the production of
molecular oxygen (O2) as byproduct. It would be hard to overemphasize the importance
of O2 production to the plants, much less its importance to all the other organisms (such
as ourselves) that require O2. Consequently, O2 production is widely assumed to be an
essential, or obligate, aspect of photosynthetic metabolism. NOT!
Well, then, what is the basic essence of photosynthesis?
The essence of photosynthetic energy metabolism is an electron transport chain driven
by light energy captured by chlorophyll. Water is the ultimate source of the electrons
used by green plants in their electron transport chains. Releasing those electron from
water by chemical oxidation produces O2 as a by product (actually, a waste product). In
fact, many photosynthetic bacteria do not use water as the source of electrons for their
electron transport chains, and therefore they do not produce O2. This is referred to as
anoxygenic photosynthesis to distinguish it from the oxygenic photosynthesis of green
plants.
OK, so not all photosynthetic organisms produce O2. Why would I think that is more
than a mildly interesting bit of biological trivia?
Because, startling as it may be, oxygenic photosynthesis evolved relatively recently in
the history of life on earth. Anaerobic photosynthesis existed on earth for billions of
years before the evolution of oxygenic photosynthesis.
That doesn’t make any sense. How could there have been oxygen in the atmosphere
for other organisms, like animals to use?
Apparently there wasn’t. For at least 2 billion years after the origin of life the earth was
anaerobic.
Oh. So, the evolution of oxygenic photosynthesis worked a profound revolution in the
chemistry of the earth’s atmosphere?
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Lab 5. Photosystem II
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Yes, the last 2 billion years or so the planet has been aerobic. All aerobic organisms,
including animals have arisen since then from anaerobic ancestors.
Well, golly, how did photosynthetic organisms manage to begin using water as the
source of electrons?
Briefly put, this required the evolution of a second electron transport chain, kind of a
twin to their original one except that the first component of the chain the so-called prs
(chlorophyll) was capable of oxidizing water. In modern biology we refer to this second,
O2 evolving prs as PSII.
Aha, that what that “Z Scheme” business was all about that they tried to teach us in
Intro. Biology. Could you go over that again.
Yes, that is what the Z scheme is all about, and no, we do not have the wherewithal to
review the details here. Please consult the reading for that.
Suffice it to say that the reaction center of Photosystem II is a complex, "solid-state"
system of proteins and electron carriers that shuttle electrons from an external source,
water, to a small organic molecule (a quinone) that diffuses away from the reaction
center. The continued operation of this process depends on energy input from light
because water is a very poor electron donor relative to the quinone acceptor. In fact,
without light energy , electrons would tend to flow spontaneously in the reverse direction
(i.e. from quinones to oxygen).
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Lab 5. Photosystem II
Quinone (Oxidized Form)
O
CH 3
CH 3
+2
CH 3
(CH 2
C
CH
CH 2 ) 9
H
+2
O
2 e- + 2 H +
OH
CH 3
CH 3
+1
(CH 2
C
CH
CH 2 )9
H
+1
CH 3
QuinoneH 2 (Reduced Form = "Hydroquinone")
OH
The overall process carried out by PS II could be summarized as:
Δ 4 electrons
2 H2O + 2 QUINONE B Ox + 4 H +out + 4 hv
O2 + 4 H+in + 2 QUINONE B Red
Notice that protons (H+) do not "cancel out" of the reaction because the H+ produced by
oxidation of water appear inside the thylakoid, while those consumed by reduction of the
quinone come from the stroma. This contributes to the electrochemical gradient used to
drive ATP synthesis in the chloroplast.
One more thing. What does all this have to do with our experiment?
The experiment uses a biochemical “trick” (the Hill Reaction) to measure the electron
flow through Photosystem II. This could also be accomplished by measuring O2
production. However, measuring O2 production requires special instrumentation. With
our biochemical system we can simply use our spectrophotometers. So, the
experiment is about studying properties of Photosystem II.
Hill’s Reaction; Photoreduction of an Artificial Electron Acceptor.
In 1937 Robert Hill provided the first evidence that photosynthesis depends on separate
light-requiring and light-independent reactions. In Hill’s case, a well characterized,
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Lab 5. Photosystem II
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artificial electron acceptor with a redox potential similar to a quinone was available. No
inventions were apparently necessary on this occasion, just the novel idea of using the
electron acceptor in an illuminated chloroplast suspension. The artificial acceptor is
actually a dye (dichlorophenol indophenol: DCPIP) and is sometimes referred to as the
"Hill Reagent". DCPIP changes from blue to colorless upon reduction. The introduction
of DCPIP into a chloroplast preparation interrupts the passage of electrons between
photosystem II and photosystem I, apparently by accepting electrons from the reduced
quinone after it leaves the PSII complex. The loss of the blue color of a DCPIP solution
is proportional to the extent of DCPIP reduction and can be measured with a
colorimeter.
Using the artificial electron acceptor trick, Hill not only demonstrated light-dependent
photoreduction, but also created a powerful experimental system for quantitative
investigations of photosynthesis. The Hill reaction does not measure photosynthesis per
se. but photoreduction of DCPIP by electron transport through PSII. Understand this
point before proceeding. How might you measure photosynthesis in a more global way?
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Lab 5. Photosystem II
Experimental
Work in groups of 4.
Be sure you have graph paper in lab to plot data points as you obtain them.
There are 5 experimental subsections. Subsections A and B must be completed first.
Subsections C, D and E may be done in any order.
You may not have time to complete all subsections today. It would be better to work
carefully and obtain reliable data than to try rushing through everything generating
random numbers.
A.
B.
C.
D.
E.
Qualitative demonstration of DCPIP photoreduction.
Demonstrating saturation of chloroplasts by DCPIP.
Effect of light intensity
Effect of temperature
Effect of wavelength
Hazard Information
!
DCPIP is toxic.
!
Wear gloves while conducting the following procedures.
!
Clean up any spills containing DCPIP immediately.
!
Solid waste (gloves, tissues, cuvettes, pipette tips) goes in the labeled bag at your
bench.
!
All DCPIP-containing liquid is poured into a waste beaker at your bench. Glass tubes
should be rinsed with a small amount of di H2O and then placed (without tape) in the
labeled pan provided in the hood.
! At the end of lab you empty the liquid waste beaker into the large labeled waste
bottle in the hood.
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A. Qualitative demonstration of DCPIP photoreduction.
This initial section simply shows you how to set up the Hill reaction and demonstrates
qualitatively that it works as advertised.
1. The "standard reaction" consists of:
Reaction buffer (RB)
DCPIP ( 4.0 mM)
Chloroplast suspension
1.0 ml
30 µl [subject to adjustment, see below]
10 - 20 µl [subject to adjustment, see below]
The contents of the tube are mixed by covering with clean parafilm and inverting.
The room lights will be turned off, so the reaction “officially” begins each time you put
the tube in front of the fluorescent lights on the side bench. Time spent away from
the light doesn’t count. The total reaction time therefore is the sum of the time
intervals the tube spends in the light.
The chloroplast suspension is prepared from fresh spinach. The procedure followed
is posted on the web site. We have experienced problems with debris in the
suspension plugging the orifice of the micropipette tips, leading to grossly erroneous
measurements. To mitigate this problem, enlarge the tip orifice by excising a
millimeter or two from the tip with a razor blade. Remember that razor blades should
only be discarded in the red plastic sharps disposal containers, never the ordinary
trash.
Keep the chloroplast stock suspension on ice.
RB is hypoosmotic relative to the chloroplast stroma. Therefore the chloroplasts will
rupture by osmotic lysis. This allows the DCPIP direct access to the thylakoid
membranes.
2. Spectrophotometer setup.
Set the wavelength to 600 nm and be sure to check the filter selection.
Zero the colorimeter using a cuvette containing RB or water.
Use plastic microcuvettes for all spectrophotometer readings.
3. Set up a large tube, “Tube A” with 1.0 ml of RB and 0.030 ml of DCPIP (see table
below).
Read the absorbance of tube A.
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Lab 5. Photosystem II
Based on the absorbance value you obtain, adjust the volumes of RB and DCPIP, if
necessary in the next step (Step 4.), so that the absorbance is 1.3 – 1.5. The total,
volume should remain 1.03 ml. This adjustment will maximize the number of
readings falling the range of values that can be reliably measured by your
instrument.
4.
To demonstrate DCPIP photoreduction, set up the following series of 5 small tubes
(A-E) according to the table below:
Tube
A
B
C
D
E
RB*
(ml)
1.0
1.0
1.0
1.0
1.0
DCPIP*
(ml)
0.030
0.030
0.030
0.030
Chloroplasts
(ml)
0.01
0.01
0.01
Light
+
+
+
Volumes may be adjusted as described in Step 3.
Mix the chloroplast suspension well immediately before pipetting. Be sure your tip is
not plugging up as you pipette. You may need to enlarge the orifice of the tip slightly
by cutting 1-2 mm off the end.
Tube E is the “experimental” and the other 4 are “controls”. What are they
“controlling for”?
5. Mix the tubes thoroughly.
6. Wrap tubes B and D in aluminum foil to completely exclude light.
7. Place all 5 tubes in front of the lights at the position where they are exposed to 400
foot-candles. (Note that it is advisable to use the same light meter throughout the
lab.)
8. Illuminate the tubes till you notice that the DCPIP in tube E has been significantly
reduced. This should not take longer than a couple minutes.
7. Visually examine all tubes and confirm that the results are consistent with your
expectations.
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9. Transfer 1.0 ml samples from each tube to separate microfuge tubes and spin them
in a microcentrifuge for 1 minute to pellet the chloroplasts and cell debris.
Remember to balance the rotor.
To balance 3 tubes in the rotor, place them at an angle of 120° from each other.
10. Read the A600 of the clarified supernatants. Be careful not to suck up cell debris
when you transfer the sample to the cuvette.
Stop to consider the following points.
•
Was there any non-biological photoreduction of DCPIP? This will be manifest in
tube A by comparing the A600 reading before and after illumination and by
comparing tube B with tube A.
•
Is the visible disappearance of blue color in tube E consistent with its Δ A600?
•
Did the chloroplasts reduce DCPIP in the dark? This would be evident by
comparing tube D with tube E.
Have the instructor review your results for this section and be sure you understand
them before proceeding.
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Lab 5. Photosystem II
B. Demonstrating saturation of chloroplasts by DCPIP.
The appropriate volume of chloroplast suspension may vary from day to day with the
quality of spinach and vagaries of the chloroplast isolation. You need sufficient
chloroplast suspension to produce measurable dye reduction in a reasonable time
but not so much that the dye reduction rate is limited by DCPIP rather than by
chloroplasts. This is another way of saying, "If you have too many chloroplasts, they
will not be operating at Vmax."
1. Set up a single reaction in a LARGE tube by scaling up the volumes of RB and
DCPIP 10-fold. Your total reaction volume is 10.3 ml rather than 1.03 ml.
Include any adjustment in the amounts of RB and DCPIP shown necessary in
part A.
2. Add 0.1 ml of chloroplasts, mix, and then transfer a 1.0 ml sample to a microfuge
tube. Spin the 1 ml sample in the microfuge tube for 1 minute to pellet debris and
then determine A600 of the supernatant. This is your zero-time reading.
3. Place the large tube in the light at an intensity of 400 foot-candles.
4. Remove similar 1.0 ml samples from the large tube at intervals of 3 or 4 minutes,
spin them and then read A600 of the supernatants. Mix the large reaction tube by
inversion (parafilm !) just before transferring each sample. It is important to
record the time that the sample was taken, and to measure the absorbance as
soon as possible. advisable to Continue until all the solution in the large reaction
tube is gone. (9 samples X 3-4 minutes).
5. Draw a graph of A600 vs. time in minutes.
We will accept as evidence of Vmax (saturation) a LINEAR change in
absorbance values (with time). This shows that altering the substrate (DCPIP)
concentration does not affect the rate of the reaction.
If your decrease is not constant (i.e. NOT linear) then it may mean the
chloroplasts are not saturated. In this case, use less of the chloroplast
suspension and repeat the reaction. You must demonstrate a linear decrease
before proceeding the following sections of the experiment.
6. Calculate the rate of DCPIP reduction as Δ A600 / minute and then convert to
millimoles DCPIP / minute.
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Lab 5. Photosystem II
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C. Effect of light intensity
What is the rate limiting factor in our experimental system? How might you
experimentally address this point? You might start by asking if light is the limiting factor.
If it is, there should be a linear relationship between light intensity and photoreduction
rate. This is analogous to considering photons as a "substrate" for the photosystem. A
linear relationship between intensity and rate suggest that light is limiting even at the
highest intensities. A non-linear relationship might indicate that some other component
(DCPIP) is limiting or that light is damaging the photosystem.
Set up 4 tubes using the same volumes as in section B. Expose the tubes to different
light intensities and measure the DCPIP reduction rates exactly as you did in B.
Intensities of 400 fc, 200 fc, 100 fc and 50 fc would do nicely.
For this, and subsequent experiments, once a particular reaction has "bottomed out":
(i.e. is no longer decreasing) there is no need to continue taking time points.
Draw a “best-fit” straight line to graph your initial data points vs. time, and use the slope
to calculate the rate of dye reduction (millimoles DCPIP/minute).
Then, create a summary graph of the RATE of dye reduction vs. the light INTENSITY.
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Lab 5. Photosystem II
D. Effect of temperature
Chemical reactions exhibit a strong and characteristic response to temperature. By the
1920's chemists had collected enough measurements to allow Nernst to formulate an
empirical generalization ...
"A rise of 10° C usually doubles or trebles the velocity of a reaction..."
Enzyme-catalyzed biochemical reactions show a similar temperature response, at least
for the range of temperatures wherein the enzyme retains its active conformation.
Test the effect of temperature on DCPIP reduction to see if it is consistent with this
generalization.
Prepare identical reaction tubes, one for each temperature, using the volumes from
section B. Chill one tube by placing it in a beaker of ice water (0°C). Place the second
tube in a beaker of water at room temperature. Additional tubes can be incubated in
pre-warmed water. (Measure and record all temperatures in your notebook.)
Be sure all the beakers are at the same distance from the light and that they are close
enough to the light to achieve a reasonably fast reaction at room temperature. Make
sure that the tubes are positioned at the sides of the beakers so that light won’t be
blocked by the ice. Occasionally wipe the condensation from the iced beaker.
Measure DCPIP reduction rate exactly as you did in section B by spinning 1.0 ml
samples and then determining A600. Take samples at intervals of 3-4 minutes for a total
of 8-10 samples for each temperature.
The heat tolerance of the reaction centers is not a trivial ecological concern. After all,
leaves are designed to expose the reaction centers to sunlight, which may be direct,
intense and which may lead to heating.
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Lab 5. Photosystem II
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E. Effect of wavelength
An “action spectrum” is a graphical representation of the magnitude of a biological effect
(dye reduction rate in this case) as a function of wavelength. We cannot produce a bona
fide action spectrum in this sense because we do not have the technical capability of
exposing our samples to monochromatic beams of light of sufficient intensity to produce
a measurable response. Instead, we will take the admittedly crude approach of using
colored plastic to expose samples to subsections of the visible spectrum, though this is
not the ideal way to produce specific wavelengths.
Holding all conditions except illumination color constant, compare the dye reduction
rates in response to illumination by 4 different colors. You will need, once again, to set
up tubes with the volumes prescribed in section B and read samples following the same
standard procedure.
Set the tubes as close as possible to the light source in order to get the maximum rate
of photoreduction. Measure the light intensity in each box so that you can correct
(normalize) for the different intensity by dividing the measured rate by the intensity. This
gives a rate per foot-candle and allows you to compare the effectiveness of the light
based on color alone.
Calculate the rate of DCPIP reduction for each color as Δ A600 / minute and then
convert to mM DCPIP / minute.
The transmission spectrum for each filter is available on the web.
5.12