Data Analysis - The Hill Reaction in Isolated Chloroplasts
INTRODUCTION
This exercise contains data from an experiment that investigates one of the
important processes in photosynthesis called the Hill reaction. Your assignment is to
present and analyze the data as Results and Discussion sections of a lab report.
Photosynthesis is the process by which plants convert light energy to chemical energy.
The familiar equation:
light
nCO2 + 2nH2O
→
(CH2O)n + nO2 + nH2O
chloroplasts
summarizes the process but gives little indication of its complexity. There are actually
two sets of reactions, the light-dependent and the light-independent reactions. As their
names imply, one reaction series requires light, while the other does not. The products
of the light reactions are molecular oxygen (O2), ATP, and reduced electron carrier
(NADP+). The ATP and reduced carrier are used in the dark reactions to convert carbon
dioxide to carbohydrates. This exercise examines the Hill reaction, an important phase
of the light reactions. The Hill reaction is the transfer of electrons from water to an
electron acceptor in the presence of light and chloroplasts. This lab will follow the rate
of the Hill reaction under several experimental conditions.
In 1937, Robert Hill showed that isolated chloroplasts can evolve oxygen in the
absence of CO2. This finding was one of the first indications that the source of the
electrons in the light reactions is water. In his in vitro system, Hill provided an artificial
electron acceptor. The artificial acceptor intercepts the electrons after Photsystem II (PS
II) but before they reach Photosystem I (PS I). The path of electrons from water to the
artificial acceptor (A) is, thus:
Various dyes can be used as the artificial electron acceptor (A) so that a general
equation, known as the Hill reaction, can be written:
H2 O + A
light
→
AH2 + 1/2O2
chloroplasts
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The Hill reaction is formally defined as the photoreduction of an electron acceptor
by the hydrogens of water, with the evolution of oxygen. In vivo, the final electron
acceptor in the light reactions is NADP+.
In isolated chloroplasts, a convenient method for measuring the rate of the Hill
reaction is to use a dye that changes color as it is reduced. These experiments used the
dye 2,6-dichlorophenolindophenol (DCIP), which is blue in its oxidized form and
colorless in its reduced form. The change in absorbance, which is measured at 600 nm,
was used to measure the rate of the Hill reaction under a variety of conditions in two
experiments.
Experiment 1: Effect of Inhibitor on the Hill Reaction
In the first experiment, the normal rate of the Hill reaction was measured and
compared to the rate in the presence of an inhibitor. The inhibitor was 3-(3,4
dichlorophenyl)-1, 1-dimethylurea (DCMU), an herbicide. DCMU blocks both electron
transport and phosphorylation by interrupting electron flow at the beginning of the major
electron transport chain. The control in Experiment 1 was a reaction mixture kept in the
dark.
Experiment 2: Effect of Light Intensity on the Hill Reaction
The second experiment investigated the effect of light intensity on the rate of the
Hill reaction. It varied the light intensity by placing the reaction vessel at different
distances from the light source. Keep in mind that light intensity decreases as the
square of the distance from the source.
Chloroplast Isolation
The chloroplasts were obtained from spinach leaves using a modification of a
standard fractionation procedure (Whatley and Arnon, 1962). After homogenization in a
mortar and pestle with a buffered, isotonic salt solution and a small quantity of sand, the
suspension was centrifuged at 1400 RPM for 1 min. The brief centrifugation at low RCF
sediments debris and whole cells. The supernatant was then spun at 3500 RPM for 10
min, sedimenting the chloroplasts (and nuclei). The supernatant was discarded and
the pellet was resuspended in 10 ml of ice-cold NaCl-buffer.
Four ml of the chloroplast suspension was added to a clean, chilled test tube and
diluted with 6 ml of ice-cold NaCl-buffer. This was the diluted chloroplast suspension
that was used to measure the Hill reaction.
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Experiment 1: Effect of Inhibitor on the Hill Reaction (25 cm from light)
Table 1. The experimental set up.
Tube
NaClbuffer*
Blank
1*
2
3
3.5 ml
3.5 ml
3.5 ml
3.0 ml
DCIP
(4 x 10-4 M)
DCMU
(2 x 10-4 M)
--0.5 ml
0.5 ml
0.5 ml
------0.5 ml
Distilled
water
Chloroplast
suspension*
0.5 ml
-------
1.0 ml
1.0 ml
1.0 ml
1.0 ml
Tube 1 was wrapped with two layers of aluminum foil so that no light could enter. Its
absorbance was measured after 10 min. The reference blank was used to calibrate the
spectrophotometer. Tube 2 contained the normal reaction and Tube 3 included the
inhibitor.
Tube 2 was mixed and the absorbance was immediately measured (time 0). Then
absorbance readings were taken at 1-min intervals up to 10 min. Tube 3 was then
mixed and then absorbance readings were taken at 1-min intervals up to 10 min.
Results
Table 2. Absorbance values for normal reaction at 25 cm without inhibitor.
Time (min)
0
1
2
3
4
5
6
7
8
9
10
Group 1
0.83
0.70
0.61
0.53
0.49
0.44
0.40
0.38
0.36
0.33
0.30
Group 2
Group 3
0.90
0.75
0.65
0.57
0.51
0.48
0.42
0.40
0.39
0.35
0.32
0.79
0.69
0.58
0.51
0.47
0.42
0.38
0.35
0.33
0.30
0.27
3
Group 4
0.86
0.71
0.63
0.55
0.51
0.43
0.41
0.37
0.35
0.34
0.28
Table 3. Absorbance values for reaction at 25 cm with inhibitor. Time (min) Group 1 0
1
2
3
4
5
6
7
8
9
10
0.90
0.89
0.88
0.87
0.88
0.87
0.88
0.87
0.87
0.88
0.88
Group 2 Group 3 Group 4 0.95
0.94
0.95
0.95
0.94
0.94
0.94
0.94
0.94
0.94
0.94
0.83
0.83
0.83
0.82
0.82
0.82
0.82
0.82
0.82
0.82
0.82
0.92
0.92
0.92
0.92
0.91
0.91
0.91
0.91
0.91
0.91
0.91
Table 4. Absorbance values for the dark tubes.
Time (min)
Group 1
Group 2
Group 3
Group 4
0.82
0.91
0.80
0.87
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Experiment 2: Effect of Light Intensity on the Hill Reaction
This experiment measured the normal rate of the Hill Reaction at two different light
intensities (12 cm and 50 cm from the lamp).
Results
Table 5. Absorbance values for normal reaction without inhibitor at 12 cm.
Time (min)
0
1
2
3
4
5
6
7
8
9
10
Group 1
0.83
0.70
0.61
0.53
0.49
0.44
0.40
0.37
0.33
0.29
0.27
Group 2
Group 3
0.90
0.75
0.65
0.57
0.51
0.48
0.42
0.40
0.39
0.35
0.32
0.79
0.69
0.58
0.51
0.47
0.42
0.38
0.35
0.33
0.30
0.27
Group 4
0.86
0.71
0.63
0.55
0.51
0.43
0.41
0.37
0.35
0.34
0.28
Table 6. Absorbance values for normal reaction without inhibitor at 50 cm.
Time (min)
0
1
2
3
4
5
6
7
8
9
10
Group 1
0.70
0.67
0.66
0.65
0.64
0.63
0.62
0.62
0.61
0.61
0.59
Group 2
0.90
0.87
0.86
0.85
0.84
0.83
0.82
0.82
0.81
0.81
0.79
Group 3
0.79
0.76
0.74
0.70
0.64
0.62
0.60
0.59
0.57
0.55
0.53
5
Group 4
0.86
0.81
0.80
0.79
0.78
0.75
0.74
0.72
0.70
0.67
0.65
Data Analysis and Questions for Discussion
Write a Results section presenting the data in tables and figures and describing relevant
trends in paragraph form. A results section is not just tables and graphs but includes a
written description of the data. Include the following data analysis (1-3) and address
items 4-7 in your discussion section in paragraph form. Do not write the answers as a
list.
1. Average the data for each time for each experiment. Then determine the absolute
value of the change in absorbance (ΔA) at each time interval, i.e., the absolute
value of the difference between the average initial absorbance at time 0 and
the average absorbance reading at each specified time.
2. Plot the rate of the Hill reaction for tubes with and without the DCMU (Experiment 1)
on a single graph. Plot the change in absorbance (ΔA) versus time. Include the
origin as a point for all curves, i.e., the 0-min value for ΔA is assumed to be 0.
3. Plot the rate of the Hill reaction for the tubes in Experiment 2 on a single graph. Plot
the change in absorbance (ΔA) versus time. Include the origin as a point for all
curves, i.e., the 0-min value for ΔA is assumed to be 0. Include the data for tube 2 in
experiment 1 on this graph. Label each plot with the distance from the reaction
vessel to the light source.
4. Describe and explain the patterns observed in the curve for the tube with the
herbicide.
5. In which tube in Experiment 1 does the reaction proceed most rapidly? Explain.
6. Discuss the relationship between the rate of the Hill reaction and the light intensity.
Include in you discussion any possible sources of error in Experiment 2.
7. Discuss the location and significance of chloroplast reactions demonstrated in the in
vitro Hill reaction.
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