Bio 122L (Laboratory for Bio 322) Exercise The Hill reaction: Effect ...

Fall 2003
David Herrin, Instructor
Introduction
Bio 122L (Laboratory for Bio 322) Exercise
The Hill reaction: Effect of a Herbicide and Pronase
R. Hill first demonstrated that a thylakoid membrane preparation could evolve O 2
without CO 2 being present. The reaction required light and a suitable hydrogen (i.e.,
electron) acceptor. The success of this experiment showed that oxygen is evolved by
the light reactions, and also indicated that water was the electron donor. Today, the Hill
reaction is often referred to as photolysis (i.e., light-dependent splitting) of water with the
subsequent evolution of oxygen. However, it is frequently assayed using an electron
acceptor such as 2,6-dichlorophenol indolephenol (DCIP) that changes color when it is
reduced (see below) rather than assaying the release of oxygen, which is more difficult
to quantify. The reaction we will use to measure the Hill reaction is the following:
DCIP (blue) + H 2 O -----> DCIP-H 2 (colorless) + 1/2 O 2
Since half an oxygen molecule would not be released, a more physiological version is:
2DCIP (blue) + 2H 2 0 -----> 2DCIP-H 2 (colorless) + O 2
Thus, with this assay, the extent of the Hill reaction is a function of the loss of blue
color, which is measured as the loss of absorbance at 620 nm. The Hill reaction
assayed in this way is mostly a measure of the light-dependent activity of Photosystem
II, since DCIP accepts electrons from the electron transport chain before Photosystem I,
probably from the cytochrome f/b 6 complex.
In this lab you will see how this reaction, still commonly used to measure
Photosystem II, is assayed, and you will look at the effect of a herbicide (Diuron) and a
protease (pronase) on the electron flow from PSII. You will use isolated membranes
from spinach that consist mainly of thylakoid membrane vesicles enriched in PSII.
These vesicles are large fragments of stacked membranes that close up as vesicles
when the membranes are fragmented during homogenization. These vesicles (from the
stacked regions) are very large, and sediment rapidly in a centrifuge. We will take
advantage of this and isolate the membranes by differential centrifugation. It should be
noted that thylakoid membranes isolated in this way are still right-side out, i.e., the
stromal face of the membrane bilayer faces out as it does in vivo.
Diuron, also known as DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is one of a
family of compounds that have been used extensively as herbicides. However, they
have fallen out of favor lately, because they are very stable and persist in the
environment. These compounds are purported to be potent inhibitors of photosynthesis,
probably acting at the Quinone b site.
Pronase is a non-specific protease that is isolated from a bacterium. It degrades
proteins at a number of different peptide bond combinations (e.g. aspartic-glutamate,
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tryptophan-proline, etc.). Proteases can be used to probe the structure and function of
membrane-associated proteins; since most proteases (like pronase) are soluble
enzymes, they can only degrade those portions of membrane proteins that stick out of
the membrane. The membrane-embedded portions, and those portions of the proteins
that stick out on the opposite side of the vesicle membrane from the protease (i.e., the
thylakoid lumen, in this case) are protected from degradation. Thus, we will use pronase
to find out if there are any important regions of the proteins that mediate the Hill reaction
(i.e., mainly Photosystem II proteins) that project away from the membrane on the
stromal side.
Protocol
A. Preparation of thylakoid membranes
1. Before beginning, place a mortar and pestle, a 15 ml centrifuge tube, and, if possible,
a filtration flask on ice. You want to keep the plant material, and enriched
membrane fraction cold to minimize proteolysis.
2. Weigh out ~3 g of fresh spinach (which was kept cold until use) on the top-loading
electronic balance.
3. Shred the leaves with scissors into small pieces, and then homogenize the leaf
pieces with the mortar and pestle, after adding 5 ml of buffer (Tricine, 0.05 M pH
6.5). After grinding for ~1 minute, add another 5 ml of buffer and grind for another
minute.
4. Line a buchner funnel with 2 Kimwipes, and then filter the green homogenate through
it into a filter flask, using gentle vacuum if necessary. Pour the filtrate into a 15 ml
centrifuge tube, and discard the residue/Kimwipes.
5. Centrifuge the green filtrate for ~5 min in a tabletop centrifuge at high speed; a green
pellet should be apparent. Carefully pour off the supernatant (you want the pellet)
and discard it.
6. To the pellet, add ~3 ml (accuracy is not critical here) of cold Tricine buffer and
resuspend this membrane fraction by vortexing (keep tube on ice AMAP).
7. Centrifuge the crude membrane fraction again for ~5 min, pour off the supernatant,
and then resuspend the pellet in ~3 ml of cold Tricine buffer with vortexing. This
fraction contains mostly stacked thylakoid membranes.
B. The Hill Reaction
1. Place a 400 ml beaker filled with tap water (room temperature) adjacent to a light
source (one or two microscope lamps should do).
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2. Label three 10-15 ml, clear, glass test tubes (labels of 1,2,3 will do).
3. Under subdued light (if possible), pipet 1.25 ml of DCIP (1.1 x 10 -4 M) into each tube.
4. Then pipet 2.5 ml of Tricine buffer into each tube.
6. Double-wrap one of the tubes with aluminum foil so that light cannot enter. Then
place all three tubes in the water bath (beaker) in the path of the light source for two
minutes (this is to pre-warm the reaction mixtures).
7. Right before pipetting the membranes, vortex your thylakoid preparation (to get it as
homogeneous as possible), and then add 0.25 ml of the membrane suspension to
each pre-incubated tube. Mix by quick vortexing, and place the tubes back into the
water bath close to the light source.
8. After 5 min, measure the absorbance (at 620 nm) of the mixture that had been
wrapped in aluminum foil, and from one of the mixtures that had been exposed to
light. You can use water as a blank to zero the machine (to 100% transmittance or 0
absorbance).
Note: To read the absorbance of the mixtures, quickly pour the contents into a cuvette,
insert the cuvette into the machine, and then close the lid. Read the absorbance. Then
pour the mixture back into the original tube (in case you need it again). After reading the
dark-incubated tube, add a few grains (a pinch) of ascorbic acid to the tube. Notice what
happens to the solution.
6. After another 5 min (~10 min total) have elapsed, take an absorbance reading (at 620
nm) of the other (#3) light-incubated tube.
7. The difference between the absorbance values of the dark and light-incubated tubes
will give you an indication of the extent of the Hill reaction during the incubation.
C. Effect of Diuron and Pronase on the Hill Reaction
1. Label four more tubes 1- 4.
2. The additions to make to each tube are described in the Table below. One tube will
be the dark-incubated control, and the other 3 will be put in the light. Diuron will be
added to one of the light-incubated tubes, and pronase to another one. After 10
minutes, read the absorbance at 620 nm for each tube, to determine the effects of
the treatments on the Hill reaction.
Note: Be sure to vortex the membrane suspension right before pipetting it, because the
membranes tend to settle out.
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TABLE 1 - EFFECT OF DIURON AND PRONASE ON THE HILL REACTION
All the volumes below are in ml. Add the components in the order listed.
Tube Number
Components 1 2 3 4
Tricine Buffer 2.50 2.50 2.40 2.40
Thylakoids 0.25 0.25 0.25 0.25
Diuron (2 mM) 0.00 0.00 0.10 0.00
Pronase (4 mg/ml) 0.00 0.00 0.00 0.10
3. Vortex the tubes to mix the reactions, and then incubate for 5 min at room
temperature (to give the pronase a chance to work).
4. Wrap tube #1 in aluminum foil.
5. Then add the DCIP to the tubes as follows:
DCIP 1.25 1.25 1.25 1.25
Total Vol. 4.00 4.00 4.00 4.00
Light (minutes) none 10 10 10
Abs. @ 620nm
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The Report
A. Objectives and general protocol
Briefly describe the objectives of the experiment and how it was performed. This part
should be no more than 250 words.
B. Results
Then report the data as follows:
1. Construct a table of your absorbance readings for the different treatments. Calculate
the ∆A (i.e., change in absorbance compared to the dark control) for each
experimental sample, and include the values in the table.
2. What do the changes in absorbance of the reaction mixtures reflect? Is it lightdependent?
What is being measured here?
3. Construct a bar graph of the Hill reaction activity (i.e., ∆A) for each of the treatments
in part C.
C. Conclusions and Discussion (or questions to be addressed)
1. What was the effect of Diuron on the Hill reaction in spinach? Could this result
explain the herbicidal properties of Diuron?
2. What was the effect of pronase on the Hill reaction? What does this indicate about
photosystem II (or some component of PSII)?
3. Abscorbic acid is often used as an antioxidant in food, and in photosynthesis
research as an electron donor. What was the effect of adding abscorbic acid to the
reaction tube in part B? What must it have done to the DCIP? What do you think the
purpose was of doing this test?
4. Indicate why you think you are measuring thylakoid PSII activity in these assays as
opposed to a contaminating redox system from the endoplasmic reticulum or
mitochondria.
5. The PSII protein that binds the terminal electron acceptor of PSII, the Qb quinone, is
known to be particularly sensitive to proteases. Could the result you obtained with
the pronase treatment be explained by assuming that pronase damaged this
protein? Can you suggest a way to determine which PSII protein(s) were damaged
by pronase?
6. Do you think Diuron would make a good herbicide? Keep in mind that with any
herbicide, or pesticide, etc., you want the drug to be as specific as possible.
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