Biology 3460 – Week 5
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Biology 3460 - Plant Physiology - Lab Exercise 6
Chlorophyll Content
Objectives:
This lab is intended to:
(1) review the process of photosynthesis,
(2) provide an example of how the Hill reaction can be used to study photosystem II,
(3) provide practical experience at using spectrophotometry to measure the light driven evolution of
oxygen in chloroplasts isolated from spinach leaves, and
(4) provide additional practice in data analysis and scientific writing.
The reactions that are collectively known as photosynthesis occur within the chloroplast and may
be separated into two metabolic pathways, historically called the 'light' reactions and the 'dark'
reactions. These reactions occur in different regions of the chloroplast. The 'light' reactions take
place in the thylakoid membranes while the 'dark' reactions (carbon fixation) occur in the stroma.
The light reactions harvest sunlight energy using two photosystems. The absorbed light energy
is used to facilitate the transfer of electrons between a series of compounds situated in the
thylakoid membranes that serve as electron donors and acceptors. The final electron acceptor is
NADP+, which is reduced to NADPH and reducing power. The ultimate products of these
reactions are oxygen and energy in the chemical form of ATP. The energy produced in this set
of reactions is used to fuel the dark reactions that reduce carbon dioxide to carbohydrates.
R. Hill and his colleagues found that isolated fragments of chloroplasts could evolve oxygen in
the light if an oxidized compound capable of accepting electrons from water was provided. In
this exercise you will study the light reactions, and the membrane bound steps of photosynthesis
called the Hill reaction.
Part A. Chloroplast Isolation
In this exercise you will isolate chloroplasts from spinach leaves that have been refrigerated in the
dark.
Protocol:
Work as a group of four students for this part of the lab exercise.
1. Prepare an ice bath to keep mortar and pestle, 100mL beaker and graduated cylinders chilled
throughout chloroplast isolation.
2. Remove the petiole and midrib from several spinach leaves, then weigh out 10g of leaf
material. Cut the leaf fragments into small pieces in the mortar then add 50 mL of cold 0.5M
sucrose. Grind the leaves for 2 minutes to prepare a homogenate.
3. Line a funnel with four layers of cheesecloth. Pour the homogenate through the cheesecloth
layers and collect the filtrate in a chilled 100 mL beaker.
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Biology 3460 – Week 5
4. Transfer the green filtrate to two (2) chilled 15 mL centrifuge tubes. Balance the tubes (to the
nearest 100th of a gram using a balance). Your instructor will centrifuge the filtrate at 500 x g
for 5 min.
5. Transfer the supernatant to two (2) chilled centrifuge tubes and balance the tubes (use a Pasteur
pipette to transfer the supernatant to ensure the pellet is not disturbed). Your instructor will
centrifuge the samples at 1500 x g for 7 min. While the samples are being centrifuged, take time
to clean your work area and any glassware that is no longer needed.
6. Carefully discard the supernatant using a Pasteur pipette and gently resuspend the chloroplast
pellet in 10 mL of cold 0.5M sucrose. Keep the chloroplast suspension on ice.
Part B. Determining the Chlorophyll Concentration in the Chloroplast
Suspension
Pigments absorb light to different extents depending on what wavelength of the light spectrum they
are exposed to. For quantification of a pigment, the ideal wavelength to select is the peak of the
pigment’s absorption spectrum. For example, for chlorophyll a, 663 nm is generally used, but for
chlorophyll b, 645 nm is a more appropriate wavelength to choose. The greater the concentration of a
pigment in solution, the larger the proportion of light absorbed by the sample at that wavelength.
Chemists have expressed this relationship quantitatively using the Beer-Lambert Law: A = Ecd (see
step 3 for variable definitions). In this exercise, you will make a dilute chlorophyll extract from your
original chloroplast suspension and spectrophotometrically determine the chlorophyll concentration
in the original suspension.
Protocol:
Work in pairs to complete the following exercise.
1. Pipet a 1 mL aliquot of the chloroplast suspension into a 10 mL graduated cylinder and dilute
to 10 mL with 80% acetone. Cover the cylinder with parafilm and mix by inverting.
2. Prepare the spectrophotometer to read the absorbance of the diluted chlorophyll extract.
Adjust the wavelength to read 652 nm (why is this wavelength chosen?). Without a cuvette in
the machine, adjust to 0% transmittance (left-hand knob). Blank the spectrophotometer with
the reagent blank (80% acetone) to read 0 absorbance (right-hand knob). Transfer some of
your diluted chlorophyll extract to a cuvette and read the absorbance. Record the absorbance
value below. Discard the acetone chlorophyll extract in the waste beaker provided.
Chlorophyll absorbance (A652) = ______________
3. Calculate the chlorophyll content in the diluted sample using the following equation. Record
the chlorophyll concentration of the diluted sample.
A = ECd
Chlorophyll content = __________
A = observed absorbance
E = a proportionality constant (extinction
coefficient) (= 36 mL / cm)
C = chlorophyll concentration (mg / mL)
d = distance of the light path (= 1 cm)
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Biology 3460 – Week 5
4. Calculate the chlorophyll concentration in the original chloroplast suspension (undiluted) by
adjusting for the dilution factor. In order to determine the concentration of chlorophyll in the
original suspension you must multiply the chlorophyll concentration in the diluted sample by
the dilution factor.
5. Knowing the chlorophyll content of your undiluted chloroplasts, prepare 10 mL of chloroplast
suspension containing approximately 0.02 mg / mL chlorophyll by diluting an appropriate
aliquot of original chloroplast suspension with cold 0.5M sucrose. Keep this on ice.
Part C. Hill Reaction and Effects of Chlorophyll Concentration, Light
Intensity and DCMU
Unless special precautions are undertaken when chloroplasts are isolated, the stroma and its
enzymes are lost. What are the resulting implications for photosynthesis? The thylakoid
membranes, however, are still capable of electron transport and photosynthetic phosphorylation
if an appropriate electron acceptor and substrates for phosphorylation are provided. The dye
DCPIP (2-6-dichlorophenol indophenol) can be used as a substitute for the naturally occurring
terminal electron acceptor. DCPIP is blue when oxidized (quinone form) but becomes colorless
when reduced to a phenolic compound:
DCPIP + H2O
light
chlorophyll
DCPIP-H2 + 1/2 O2
In this exercise you will investigate the effect that chlorophyll concentration, light intensity, and
a herbicide, diuron (DCMU), have on the Hill reaction.
Protocol:
Work in pairs to complete this section.
1. Adjust the wavelength of the spectrophotometer to 600nm then blank the machine with 0.1M
phosphate buffer. (Refer to Step 2 in Part B if required).
2. Label six cuvettes and add the appropriate volumes of phosphate buffer, DCPIP, DCMU, and
0.02mg/ml chloroplast suspension as outlined in Table 1. Be sure to add the chloroplast
suspension last.
3. Once all solutions have been added to each tube invert to mix and quickly measure the
absorbance of each at time 0 and record this in Table 2.
4. Place tubes 1, 2, 3, and 5 in a test tube rack 30cm from a 100 watt light source. Place tube 4
in a rack 60 cm from the same light source. Wrap tube six in aluminum foil, leaving only the
top open for access when reading absorbance, and place in the rack with tube 4.
5. Measure the absorbance of each tube at five-minute intervals for 30 minutes. Record results
in Table 2.
6. To determine the change in absorbance over time in each tube, subtract the interval
absorbance from the initial absorbance and record these results in Table 3. Report these
results to your instructor for inclusion in the class data set.
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Biology 3460 – Week 5
Table 1. Reagent volumes used in Hill reaction investigation.
0.1 M
Phosphate
buffer (mL)
4
4
4
4
0
4
Tube #
1
2
3
4
5
6
0.05 mM
DCPIP (mL)
0.05 mM
DCMU (mL)
Chloroplast
suspension (mL)
4
4
4
4
4
4
0
0
0
0
4
0
0
0.5
1
1
1
1
7. Discard the contents of all tubes in the waste bottle marked 'DCPIP waste' and clean up your
work area.
Table 2. Absorbances (A600) for the treatments investigating the effect of chlorophyll
concentration, light intensity, and DCMU on the Hill reaction.
Tube #
0 min
5 min
10 min
15 min
20 min
25 min
30 min
1
2
3
4
5
6
Table 3. Change in absorbances (A600) for the treatments investigating the effect of
chlorophyll concentration, light intensity, and DCMU on the Hill reaction.
Tube #
0 min
1
0
2
0
3
0
4
0
5
0
6
0
0 - 5 min
0 - 10 min
0 - 15 min
0 - 20 min 0 - 25 min
0 - 30 min
Biology 3460 – Week 5
Analysis:
1. Prepare a graph that shows the effect of time on change in absorbance in each of the
treatments. Means with standard deviations should be plotted.
2. Use class results and prepare three graphs that show the effect of chlorophyll concentration,
light intensity, and DCMU on the Hill reaction. Mean total change in absorbances with
standard deviations should be plotted.
3. Using a t-Test, determine if there were statistically significant differences between the
treatments. (You will need to do three different t-Tests - there are three variables that were
tested.)
4. Diuron (DCMU) belongs to a herbicide family that purportedly is a potent inhibitor of
photosynthesis, likely acting at the Qb to PQ step in the electron transport chain. Do the
results from our experiment support this hypothesized mechanism? Explain.
5. What was the effect of chlorophyll concentration on the Hill reaction? the effect of light
intensity?
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