Biology 3460 - Plant Physiology - Lab Exercise 5
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. After washing healthy spinach plant leaves (~ 30 g) in cold water, remove the petiole and
midrib, and place the leaf fragments on ice. During subsequent steps in the chloroplast
isolation, keep the leaf material, glassware, and all solutions on ice.
2. Transfer the leaf fragments to a chilled Waring blender vessel, add 100 mL of cold 'isolation'
media, and blend the mixture for 20 sec.
3. Line a funnel with four layers of cheesecloth. Pour the homogenate through the cheesecloth
layers and collect filtrate in a chilled 250 mL beaker. Discard the collected residue along
with the cheesecloth.
4. Transfer the green filtrate to two (2) chilled centrifuge tubes. Balance the tubes (to the nearest
100th of a gram using a balance). Your instructor will use the centrifuge to spin the filtrate at
500 x g for 5 min. Samples will not be spun until ALL groups have loaded their centrifuge
tubes into the centrifuge.
5. Transfer the supernatant to two (2) chilled centrifuge tubes and balance the tubes. Your
instructor will centrifuge ALL the samples at 2 000 x g for 15 min. While the samples are being
centrifuged, discard the remaining pellet from the previous low speed centrifugation.
6. Carefully discard the supernatant and gently resuspend the chloroplast pellet in 10 mL of
cold isolation medium by finger vortexing. 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
pigments 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 as a group of four students to complete the following exercise.
1. Pipet a 0.1 mL aliquot of the chloroplast suspension into a 25 mL graduated cylinder and
dilute to 25 mL with 80% acetone. Cover the cylinder with parafilm and mix by inverting
several times.
2. Measure the absorbance of the diluted chlorophyll extract at 652 nm using a
spectrophotometer. (Why is this wavelength chosen?) Adjust the wavelength to read 652 nm.
Without a cuvette in the machine, adjust to 0% transmittance (left-hand knob). Blank the
spectrophotometer with the reagent blank 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)
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 isolation medium. 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. Prepare the reagent blank (Table 1) by adding 4 mL of tricine buffer and 1 mL of isolation
media to a test tube. Cover the test tube with parafilm and gently vortex. (You only need
onee blank for every four students).
2. Adjust the wavelength to 600 nm on the spectrophotometer. Use the newly created reagent
blank to calibrate the machine. (Refer to the steps in Part B, Step 2 if required).
3. Prepare the contents of tube 1 as outline in Table 1. Vortex to mix. Measure the absorbance of
the contents of this tube at 600 nm and record the value in Table 2.
4. Label the remaining six test tubes as indicated in Table 1. Prepare the incubation mixtures
listed in Table 1 by adding the indicated amount of tricine buffer, isolation medium, DCPIP
and DCMU to each of tubes 2 through 7. Note the instructions that follow on timing of
additions of chloroplast suspension.
Table 1. Reagent volumes used in Hill reaction investigation.
Tube
No.
Tricine
buffer*
(mL)
Blank
1
2
3
4
5
6
4.0
3.0
4.0
4.0
3.0
3.0
3.0
Dil.
Chloroplast
suspension
(mL)
none
none
0.5
1
0.5
1.0
1.0
Isolation
media**
DCPIP
solution***
(mL)
DCMU
solution ****
(mL)
1.0
1.0
0.5
none
0.5
none
none
none
1.0
none
none
1.0
1.0
1.0
none
none
none
none
none
none
none
7
none
1.0
none
1.0
3.0
* Tricine buffer (0.02 M, adjusted to pH 7.5 with KOH)
** Isolation media containing 0.4 M sucrose, 0.03 M KCl and 0.02 M tricine
*** DCPIP solution (0.2 mM in tricine buffer)
**** DCMU solution (0.05 mM in tricine buffer)
5. Add 0.5 mL of the dilute chloroplast suspension (0.02 mg chlorophyll / mL) to tube 2 and 1.0
mL of the dilute chloroplast suspension to tube 3. Quickly vortex to mix. Immediately
measure the absorbance of both of these tubes at 600 nm. Record the absorbances in the
appropriate spaces in Table 2.
6. Place tubes 4, 5, and 7 in a test tube rack positioned 30 cm from a high intensity light source
(slide projector). Place tube 6 in a rack positioned 60 cm from the light source. Quickly add
0.5 mL of chloroplast solution to tube 4 and finger vortex to mix. Add 1.0 mL of the
chloroplast suspension to tubes 5, 6, and 7 and finger vortex to mix. NOTE THE TIME.
7. When tube 5 (highest chlorophyll concentration and highest light intensity) appears to have lost
most of its blue color, note the time. Quickly cover all tubes with aluminum foil and place
them on ice.
8. Measure the absorbance (to two decimal places) of each reaction mixture at 600 nm using the
previously calibrated spectrophotometer. Rinse the cuvette between samples.
9. Calculate the initial absorbances of the 6 reaction mixtures (using the data from tubes 1, 2,
and 3) and the change in absorbance after light treatment. What do the changes in light
absorbance of the reaction mixtures reflect?
10. Discard the contents of the blank and tubes 1 through 6 in the sink and flush with running
water. Discard the contents of tube 7 in the waste bottle marked 'DCMU waste'.
Table 2. Absorbances (A600) for the treatments investigating the effect of chlorophyll
concentration, light intensity, and DCMU on the Hill reaction.
Tube No.
1
No chloroplast
suspension
2
0.5 mL chloroplast
suspension, no DCPIP
3
1.0 mL chloroplast
suspension, no DCPIP
4
5
6
7
1
Tube components &
conditions
Initial
absorbance
(calculated)1
Measured
absorbance
Change in
absorbance
0.5 mL chloroplast
suspension, high-light
intensity
1.0 mL chloroplast
suspension, high-light
intensity
1.0 mL chloroplast
suspension, low-light
intensity
1.0 mL chloroplast
suspension, high-light
intensity, DCMU
For tubes containing 0.5 mL of chloroplast solution, the initial absorbance is the sum of
the measured absorbance of tubes 1 and 2. For tubes containing 1.0 mL of chloroplast
solution, the initial absorbance is the sum of the measured absorbance of tubes 1 and 3.
Analysis:
1. Use class results and prepare three graphs that show the effect of chlorophyll concentration,
light intensity, and DCMU on the Hill reaction. Mean absorbances with standard deviations
should be plotted.
Tube
No.
Tube components &
conditions
4
0.5 mL chloroplast
suspension, high-light
intensity
5
1.0 mL chloroplast
suspension, high-light
intensity
6
1.0 mL chloroplast
suspension, low-light
intensity
7
1.0 mL chloroplast
suspension, high-light
intensity, DCMU
Change in absorbance (A600)
1
2
3
4
5
6
7
8
9
10
Mean
change in
abs + s.d.
2. 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.)
3. 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.
4. What was the effect of chlorophyll concentration on the Hill reaction? The effect of light
intensity?