Thylakoid extraction
Chloroplast preparation Use Percoll instead of sucrose and test steps ahead of time (most banded
at 1.8/1.3 M sucrose interface).!
Chloroplasts are relatively large and dense, so they sediment rapidly upon centrifugation.
Consequently, chloroplasts can be separated from smaller organelles after breaking open leaf cells by
giving the brei a short spin at low speeds. Chloroplasts will be in the pellet, while most of the other
organelles will be in the supernatant. For many purposes, this is adequate purification. However,
nuclei, unbroken cells and other debris will also be in the pellet, so often further purification is needed.
Fortunately, these all have different densities from chloroplasts, so we can separate chloroplasts from
these contaminants by density gradient centrifugation. There are three ways to do this: 1) you can load
your sample suspended in low-density buffer on the top of a tube filled with medium which becomes
increasingly dense progressing down the tube. Upon centrifugation your organelles will sediment
down the tube until they reach a point where the density of the surrounding medium is equal to their
own. 2) you can prepare your sample in high-density medium, then layer media of increasingly lower
density on top of your sample. Upon centrifugation your organelles float up until they reach a point
where the density of the surrounding medium is equal to their own. 3) you can prepare your sample in a
medium which forms a density gradient upon centrifugation. Cesium chloride does this upon
ultracentrifugation, and is therefore frequently used for purifying DNA (as you will recall from the
Meselson-Stahl experiment). Silica sols such as percoll will also form density gradients upon
centrifugation. In this case you prepare your samples in such a medium then spin them long and hard
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enough for the medium to form a density gradient. Once the contents of the tube have reached
equilibrium you collect the portion of the gradient containing chloroplasts.
Unfortunately, there is no best method for preparing chloroplasts. It all depends upon what you plan to
do with them. For many purposes chloroplasts prepared by differential centrifugation are adequate,
whereas to obtain the purest fractions density gradients are the method of choice. Unfortunately, they
also take much longer, and during that time chloroplast integrity is often impaired such that light- and
CO2 -dependent photosynthetic activity may be lost.
Measuring photosynthetic activity of isolated chloroplasts
There are three basic approaches to measuring photosynthesis. The first is to measure incorporation of
CO2 into sugar. This can be done either by measuring the removal of CO2 from the atmosphere using
an instrument called an IRGA (Infra-Red Gas Analyzer) or by measuring the incorporation of
radioactive CO2 into sugar. The second approach is to measure light-dependent oxygen evolution. If
very high-quality chloroplasts are obtained one can measure light- and CO2-dependent oxygen
evolution. Frequently, chloroplast envelopes are disrupted during isolation, allowing critical stromal
components such as Calvin cycle enzymes or NADP to leak out. In this case, it is no longer possible to
measure CO2-dependent oxygen evolution. However, in such cases it is still often possible to measure
light-dependent oxygen evolution provided that another acceptor for the electrons removed from water
is provided, and numerous electron acceptors which substitute for various steps in photosynthetic
electron transport have been identified. Today we will provide our chloroplasts with ferricyanide as an
alternative electron acceptor. If all goes well they will reduce ferricyanide Fe(III)(CN)63- to
ferrocyanide Fe(II)(CN)64-.
Photosynthetic electron transport (the Hill reaction)
In 1937 Robin Hill demonstrated that isolated chloroplasts could transfer electrons to an artificial
electron acceptor and evolve oxygen in the process when they were illuminated. This showed that
electron transport and CO2 fixation (i.e. the light and light-independent reactions) are separate
reactions. We now know that what happened was that Photosystem II used light energy to remove
electrons from water and donate them to the artificial electron acceptor. The general equation for the
Hill reaction is:
light
H2O + A ----------------------> AH2 + 1/2 O2
chloroplasts
where A is an artificial electron acceptor. The specific reaction which we will measure today is:
H2O + 2 Fe(CN)6
3-
light
----------------------> 2 Fe(CN)64- + 1/2 O2
chloroplasts
THIS WEEK’S ACTIVITIES
This week we will prepare spinach chloroplasts using differential and density-gradient centrifugation.
We will grind spinach in a buffer which stabilizes and protects them, then remove large debris by
filtering them through cheese-cloth. Next we will separate them from smaller organelles by giving the
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brei a short spin at low speeds. Chloroplasts will be in the pellet, while most of the other organelles
will be in the supernatant. Unfortunately, nuclei and unbroken cells will also be in the pellet.
Therefore we will use flotation gradients to purify the chloroplasts. Next we will recover the band
containing chloroplasts and use them to measure factors affecting photosynthetic electron transport.
First we will learn how to measure the Hill reaction, and examine two chemicals known to affect it.
Recall that ordinarily electron transport is used to pump protons across the membrane to create a proton
gradient , which, in turn, is used to generate ATP. Chemicals which allow this proton gradient to be
dissipated without generating ATP are known as uncouplers, and an example of an uncoupler is
ammonia. Therefore, you will treat one of your samples with ammonia to see how this affects the rate
of the Hill reaction. Another inhibitor which you will test is DCMU ( 3-(3-4-dichlorophenyl)-1,1diethylurea). DCMU interrupts electron transport on the reducing side of PSII by inactivating one of
the electron carriers.
PREPARING CHLOROPLASTS
1.
Obtain 5 g of fresh spinach leaves from which the major veins have been removed.
2.
Cut the leaves into small pieces with a razor blade,
divide into 2 equal batches
grind each batch in a chilled mortar and pestle for 2 minutes in 10 mls ice-cold grinding buffer.


3.
Prechill and dampen four layers of cheesecloth and two layers of miracloth by pouring 10 mls
ice cold grinding buffer through it, then squeeze the brei through this filter into a 50 ml testtube kept on ice.
4.
Centrifuge 2 minutes at 2,000 x g.
5.
Discard the supernatant, and resuspend the pellet in 5 mls 60% Percoll in assay medium with a
paint brush.
6.
Carefully layer on 10 mls 40% Percoll, then 10 mls 20 % Percoll then 10 mls assay medium
7.
Centrifuge 15 minutes at 10,000 x g.
8.
Carefully remove the green band at the interface between the 40% and 20% layers using a
pasteur pipet.
9.
Transfer to a second centrifuge tube and dilute with 25 mls assay buffer.
10.
Centrifuge 2 minutes at 2,000 x g.
11.
Discard the supernatant, and resuspend the pellet in 1 ml assay buffer with a paint brush.
12.
Determine the chlorophyll concentration by mixing 50 µl of your chloroplasts with 5 mls 80%
acetone, filtering, then measuring the absorbance at 652 nm.
mg chlorophyll/ml = A652 x dilution factor
34.5
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(before starting check that the spectrophotometer is warmed up, set to 652 nm and adjusted to
0% transmittance. Use 80% acetone as your blank)
13.
Dilute your chloroplasts to 0.2 mg/ml chlorophyll using assay buffer, then keep the suspension
in the dark and on ice.
14.
Boil a 0.6 ml aliquot of chloroplasts for 5 minutes.
MEASURING THE HILL REACTION
1.
Turn off the stirrer and remove the water, then rinse with assay buffer.
2.
Add the contents of tube 1 prepared as described below.

First, add the correct volume of buffer.

Next add the correct volume of water.

Third, add the potassium ferricyanide.

Finally, add the chloroplasts.
3.
Carefully insert the oxygen electrode. Locate the groove made in the plunger and tilt the
chamber so that the groove is on the high side. Carefully push down the plunger squeezing the
bubble through the groove so that no bubbles are trapped beneath the plunger.
4.
Turn on the stirrer, waterbath and floodlamp, then once the needle has settled down read the %
oxygen saturation at 30” intervals for 5 minutes, entering the data in Table I.
5.
Remove the contents of the cuvette and rinse out with assay buffer.
6.
Add the contents of tube 2, then wrap the cuvette with aluminum foil and turn off the light.
7.
Repeat steps 3-5.
8.
Add the contents of tube 3. Note that the boiled chloroplasts are added to this cuvette.
9.
Repeat steps 3-5.
10.
Add the contents of tube 4. Note that you leave out the K3Fe(CN)6
11.
Repeat steps 3-5.
12.
Add the contents of tube 5, then turn on the stirrer, waterbath and lights. Once you the needle
settles to a steady rate measure the % saturation at 30” intervals for two minutes, entering the
data in Table I.
13.
Inject 0.5 mls of ammonium hydroxide, then once the needle settles to a steady rate measure
the % saturation at 30” intervals for three minutes, entering the data in Table I.
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14.
Remove the contents of the cuvette and rinse out with assay buffer.
15.
Add the contents of tube 6, then turn on the stirrer, waterbath and lights. Once you the needle
settles to a steady rate measure the % saturation at 30” intervals for two minutes, entering the
data in Table I.
16.
Inject 0.5 mls of DCMU, then once the needle settles to a steady rate measure the % saturation
at 30” intervals for three minutes, entering the data in Table I.
Cuvette
Buffer
water
chloroplasts
K3Fe(CN)6
1
1.0 ml
2.5 ml
0.5 ml
1.0 ml
2
1.0 ml
2.5 ml
0.5 ml
1.0 ml
3
1.0 ml
2.5 ml
0.5 ml*
1.0 ml
4
1.0 ml
3.5 ml
0.5 ml
-
5
1.0 ml
2.5 ml
0.5 ml
1.0 ml
6
1.0 ml
2.5 ml
0.5 ml
1.0 ml
* these are the boiled chloroplasts
17.
Report your findings on your datasheet. Your introduction should explain what the Hill
reaction is, why it can be measured using an artificial electron acceptor, and what you
specifically set out to do. Results should include a table showing the raw data, a graph in which
the % saturation is plotted as a function of time for tubes 1-4, and a few sentences explaining
why you performed the experiment, where the data are presented, and describing the major
trends observed. Be sure to include separate graphs for tubes 5 and 6, and state the amount
the rate changed upon adding ammonia and DCMU. Your discussion should explain the
results obtained with each tube. Be sure to explain the effects of DCMU and ammonia in
your discussion.