Hill reaction:

LAB TOPIC 7
Photosynthesis
Prelab Preparation
You should use your textbook to review the definitions of the following terms:
Photosynthesis
Biochemical reaction of photosynthesis
Photochemical reaction of photosynthesis Photosynthetic electron transport chain
Chlorophyll accessory pigments
Objectives
1. To examine carbon fixation
2. To examine electron transport in chloroplasts
3. To examine the effects of inhibitors on photosynthesis
BACKGROUND
Photosynthesis uses light energy to split H2O and harvest high-energy electrons. These energetic electrons (and
accompanying H+) are passed to CO2. In doing so, CO2 reduced to form energy-storing sugars. Cellular
respiration removes electrons from (i.e. oxidizes) sugar, captures the energy in adenosine triphosphate (ATP),
and ultimately passes the electrons to oxygen to form H2O. Organisms use the energy stored in ATP to conduct
cellular business such as transport, synthesis of biomolecules, reproduction, and sometimes cellular movement.
Remember that photosynthetic eukaryotes (plants and algae), respiration occurs as it does in animal and
fungal cells. Photosynthesis is essentially respiration operating in the opposite direction, utilizing CO2 and H2O to
produce carbohydrates and releasing O2 (Figure 7.1).
Figure 7.1
An overview of aerobic respiration and photosynthesis
EXPERIMENTAL
Carbon dioxide fixation by Elodea
When in an aqueous solution, carbon dioxide reacts
with water to form carbonic acid.
2. Fill four test tubes 3/4 full with C02-rich BTB
solution.
CO 2 + H 2O ⇔ H 2CO 3
3. Wrap 2 of the test tubes with aluminum foil,
leaving only the top open.
This results in lowering the solution's pH. As
CO2 is consumed by aquatic plants through
photosynthesis, the level of carbonic acid in a
solution will decrease, leading to an increase in pH.
Thus, monitoring pH provides an indirect measure
of the amount of CO2 consumed in photosynthesis.
This experiment uses bromothymol blue (BTB), a
pH indicator that turns yellow at pH < 6.0, green at
pH 6.0 - 7.6, and blue at pH > 7.6.
1. Place 75 ml of BTB solution into a 100 ml
beaker. Blow exhaled air through a straw into
the BTB solution until it changes from blue to
yellow-brown.
Table 7.1
4. Obtain two 2-cm sprigs of Elodea, and place one
into a wrapped tube and one into an unwrapped
tube. The other 2 tubes will have no Elodea in
them. Use these for comparison. Finish covering
the two tubes wrapped in aluminum foil.
5. Place the unwrapped test tubes directly in front
of the grow light, and the wrapped ones on your
lab bench.
6. Allow the tubes to "incubate" for 1 hour.
Proceed to the next exercise while you wait. In
Table 7.1, record any color changes that have
occurred by marking an X in the appropriate
space.
Carbon dioxide consumption by Elodea
Yellow
Green
Blue
pH < 6.0
pH 6.0 – 7.6
pH > 7.6
Elodea, light
Elodea, dark
No Elodea, light
No Elodea, dark
•
State your hypothesis regarding the experiment.
•
What are your predictions?
•
Dependent variable?
•
Independent variable?
•
Why did the BTB turn yellow as you blew through the straw?
•
What is responsible for the color change of the solutions containing the Elodea in front of the light?
•
Why might wrapped tubes with Elodea exhibit an increase in pH?
•
Why might wrapped tubes with Elodea exhibit a decrease in pH?
An overview of the light-dependent reactions of photosynthesis
In the late 1930’s, Robert Hill and colleagues observed
that under proper conditions, isolated chloroplasts retained Figure 7.2 Photosynthetic electron transport
their capacity to evolve oxygen. This phenomenon is now
known as the Hill Reaction. The Hill Reaction is part of
what are known as the photochemical reactions of
photosynthesis.
This activity is associated with
Photosystem II (Figure 7.2), in which the electrons that
originate with splitting of water are used to reduce electron
acceptors. There is a simultaneous release of oxygen. In
the intact living organism, these electrons ultimately reduce
NADP+ to form NADPH. During photosynthetic electron
transport, hydrogen ions are moved across the thylakoid
membrane as plastoquinone shuttles electrons between PS
II and the cytochrome B6/f complex. The resulting
hydrogen ion gradient is use to produce ATP. The ATP and
NADPH then are used in the biochemical reactions to
produce sugars, thus trapping light energy in the chemical bonds of carbohydrates.
Hill used artificial electron acceptors, including DCPIP (2,6-dichloroindolphenol), to trap electrons passed
through the electron transport chain from photosystem II when isolated chloroplasts are exposed to light. As the
blue, oxidized form of DCPlP becomes reduced it becomes colorless. Thus the progress of the reaction can be
monitored by the change in absorbance at 600 nm of the DCPIP solution.
DCPIP + 2H+ + 2e- →
(blue)
DCPIP-H2
(colorless)
(Remember that chloroplasts are green and so only the blue color will disappear entirely.)
The rate of the Hill Reaction is then dependent on light intensity and can be measured either as oxygen produced
or reduction of electron acceptors.
Electron Transport in Chloroplasts
1. Prepare tests tubes according to Table 7.2.
Metabolically active chloroplasts will be
provided to you. Add the DCPIP (blue dye) last.
Wrap tube #4 with foil.
source indicated by your instructor. Keep all
tube directly in the light. Do not position tube
behind one another or in front of tubes from
other groups.
2. Mix the contents of each tube by inverting each
tube several times. Place the tubes near the light
3. Observe color changes in the tubes and record
your observations.
Table 7.2
Solutions for Comparisons of Photosynthetic Reaction Rates
Tube
Chloroplasts
0.1 M phosphate
buffer pH 6.5
Water
0.2 M DCPIP
1
0.5 ml
3 ml
1.5 ml
0
2
0.5 ml
3 ml
0.5 ml
1 ml
3
0
3 ml
1.0 ml
1 ml
4
0.5 ml
3 ml
0.5 ml
1 ml
•
What were the initial and final colors of each tube?
•
What were the functions of each tube (1-4) in this exercise? Which tubes were controls?
Effects of Herbicides on Electron Transport in Chloroplasts
1. Next we will monitor the effect of a
photosynthetic herbicide on photosynthesis. We
will quantify the electron transport (DCPIP
reduction) using a spectrophotometer. Set the
wavelength for 600nm. Use water as the blank.
2. Set up tube #1 as shown in Table 7.3 and wrap it
with foil.
3. Remove the foil and take a reading of
absorbance and the place the tube back into the
foil cover.
4. Move the tube to the light source, remove the
tube and expose it to the light for 1 minute.
Cover the tube again and bring it back to get
another absorbance reading.
Record your
readings in Table 7.4.
5. Repeat steps 6 and 7 to obtain 5 minutes of
exposure to the light source.
6. Repeat steps 2-5 on a fresh tube in which you
have replaced the 0.5 ml of water with 0.5 ml of
herbicide A.
7. Repeat steps 2-5 on a fresh tube in which you
have replaced the 0.5 ml of water with 0.5 ml of
herbicide B.
Table 7.3
Solutions for Herbicide Treatments
Tube
Chloroplasts
0.1 M phosphate
buffer pH 6.5
Water or
herbicide
0.2 M DCPIP
1
0.5 ml
3 ml
0.5 ml water
1 ml
2
0.5 ml
3 ml
0.5 ml herbicide A
1 ml
3
0.5 ml
3 ml
0.5 ml herbicide B
1 ml
Table 7.4
Tube
Absorbance data
T=0
T=1 min
T=2 min
T=3 min
T=4 min
T=5 min
1
2
3
•
Plot your data on the graph paper provided on the following page.
•
What happened when you illuminated the tubes containing herbicides?
•
Base on your observations, what do can you say about the mode of action of each herbicide?
•
What other set of oxidation-reduction reactions having electron transport may have contributed to
some of the color change in the covered tube?

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