BIO 220 Laboratory Exercises
Laboratory III: The Hill Reaction of Photosynthesis
in Isolated Chloroplasts
The goal of this session is to reproduce a classic experiment1 by Dr. Robert (Robin) Hill, which
provided key evidence in describing the role of sunlight in photosynthesis. Students will be
introduced to the concept of using light energy as fuel for biological reactions.
Students will explore the Hill Reaction by:
1) observing the effect of light intensity on reaction rate
2) designing a variation of their choice, which may include:
- changing the type of light source (i.e. incandescent vs. natural or other light)
- lowering or raising pH of the reaction buffer
- effects of freeze/thaw cycles on chloroplast suspension
- altering the color (wavelength) of incident light
Introduction
Photosynthesis is a process by which light energy is converted to chemical energy. The
mechanisms by which light energy is harvested and converted to chemical energy starts with
absorption of light photons by pigments in the chloroplasts. The reaction in green plants follows:
light
CO2 + H2O
(CH2O) + O2
Shown above, CH2O stands for carbohydrate. In green plants, photosynthesis involves a series
of oxidation-reduction reactions in which water is the electron donor and oxygen is released
(Figure 1).
In the 1930s, Robin Hill investigated
electron transfer in chloroplasts using artificial
electron acceptors, such as DCIP. In his
experiments, Hill treated photons as though they
were a reactant (like succinate in the SDH
experiment). At low light intensities, the
photosynthetic rate was linear but at higher
intensities the photosynthetic rate became saturated.
In many ways photosynthesis behaves like an
enzyme-substrate reaction except that light, rather
than chemical energy, serves as the fuel.
Figure 1. Broad overview of photosystems I and II
in green plants. Reproduced from Alberts et al2.
The purpose of this experiment is to reproduce
Hill's experiment using DCIP as an electron
acceptor and to investigate the photosynthetic process through independent investigation.
Procedure
Today's laboratory has 4 phases:
1) Isolation of chloroplasts from fresh spinach leaves
2) Determination of chlorophyll concentration in the chloroplast suspension
3) Observing the effects of light intensity on the rate of the Hill Reaction
4) Performing an experiment of your choice to explore another factor affecting the Hill Reaction
Safety notice:
Acetone is a flammable organic solvent.
Acetone will MELT any type of plastic, and also parafilm.
Use only glass tubes, glass pipets, and glass cuvettes when working with acetone.
If you get concentrated acetone on your skin, flush the area generously with cold water.
Chloroplast isolation
1) Wash and weigh 25 g spinach or other green plant from which the petioles and major veins
have been removed. Cut into small pieces and place in a cold blender container.
2) Add 100 mL cold chloroplast isolation media and homogenize leaves with three 5 second
bursts at full speed. Filter the blended mixture through cheesecloth.
3) Transfer 20 mL aliquots to 50 mL centrifuge tubes and centrifuge for 5 min at 1300 x g.
Discard the supernatant and add 0.5 mL chloroplast isolation media. Mix well.
Estimation of chlorophyll concentration
1) Add 50 µL of chloroplast suspension to 5.0 mL of 90 % acetone. Cover with a small piece of
aluminum foil and vortex carefully to mix.
2) Centrifuge at 1300 x g for 5 min to pellet precipitated protein and debris.
3) Transfer approximately 3 mL of the supernatant to a glass cuvette and record the absorbance
(652 nm). The blank cuvette should contain a similar volume of a 1:100 dilution of chloroplast
isolation buffer in 90% acetone.
4) Calculate the chlorophyll concentration in mg/mL using the formula below:
5) Determine the volume of chloroplast suspension required to provide 25 µg chlorophyll to each
reaction.
Effect of photon energy on photoreduction of DCIP
1) Prepare the reaction cuvettes according to Table 1 (next page).
Notes: The final volumes in all cuvettes are identical. Each cuvette will contain slightly more
than 3000 µL. The reactant being varied is the amount of incident photons.
Cuvette #7 is reserved for independent investigation. Approve your experimental design with
your instructor before preparing cuvette #7.
2) Proceed to the instrument room.
3) Use a light meter to measure the light intensity (fc) delivered to the reaction cuvette.
Readings are approximate and should be made quickly. Your instructor will provide a
demonstration.
4) The reaction should take place in a darkened room. Chloroplast suspension (Note: DO NOT
use the chlorophyll extract) is added to the blank and placed in the reference holder of the
spectrophotometer.
5) Starting with the furthest distance (tube 4), add chloroplast suspension, mix the contents of
the cuvette, and take an initial absorbance reading at 600 nm. Illuminate the sample for 90 s
bursts, and record the absorbance after each 90 s illumination period for a total of 6 min.
Measure the absorbance of tubes 1-3 in the same way.
Note: Make sure to record the ACTUAL light intensity (fc) delivered to each sample.
Table 1. Composition of the photoreduction reaction cuvettes.
Tube
Reaction
Buffer
(mL)
DCIP**
(mL)
Light Intensity
(fc)*
0.5 mM
Stock
Target
1
2
0.5
1200-1500
2
2
0.5
1000
3
4
2
2
0.5
0.5
700-800
400-500
5
2
0.5
Dark
6
2.5
-
Blank
7
Light
Intensity
(fc)*
Actual
Chloroplast
suspension***
(µL)
DO NOT
ADD YET
Comment
This is the optimal
condition.
Reduction of DCIP
not due to
photoactivation.
Blank for tubes 1-5.
YOUR OWN
DESIGN
*A note on the use of the foot-candle (fc) as a measure of illumination (intensity of light on an object) 3: The footcandle is an English unit of measurement. The accepted SI unit is illuminance (lx or lux), which has units of lm m-2,
or lumens per square meter. Values measured in fc may be converted to lx by multiplying the fc measurement by
10.764.
** DCIP changes from blue to colorless as it becomes reduced.
*** Do not add chloroplasts until you are ready to run each reaction!!! The volume of chloroplast suspension to be
added was determined in step #5 above. This volume contains 25 µg chlorophyll.
6) Cuvette #5 (the "dark" control) will be handled differently. Add chloroplast suspension to
cuvette #5, mix, and record the initial A600 nm. Place cuvette #5 in a light-tight drawer or
cabinet (careful not to spill!), and after 6 min in the dark, record a final absorbance measurement.
Tube 5 measures the incidental reduction of DCIP, or the reduction of DCIP which results from
another process besides photoactivation.
7) Design your own experiment. Ideas include testing the photosynthetic rate in chloroplasts
from a different green plant, the effect of pH, temperature, inhibitors, different-colored light, etc.
Lab Report Write-Up Guide
Results Section
1) Prepare a graph of absorbance versus time for tubes 1-5. Include a legend which labels each
trace with the proper light intensity.
2) Use the above graph to determine Vo for each reaction, in terms of A600 nm/min. Use only
the initial linear portion of the relationship.
3) Prepare a Master Summary Table to show the effect of light intensity (fc) on photosynthetic
rate (A600 nm/min). If tube #5 demonstrated any incidental reduction of DCIP, subtract this rate
from the rates calculated for tube 1-4. Suggested column headings for this table are: Tube
number, Vo (initial velocity, A600 nm/min), and Vo (initial velocity, molecules of DCIP reduced
/min/µg chlorophyll, see below for the calculation).
4) Prepare a graph showing the effect of light intensity on reduction of DCIP (molecules of DCIP
reduced /min/µg chlorophyll). Calculation approach:
a) Convert A600 nm values to molarity of DCIP, using the molar extinction coefficient for DCIP
determined in the "Introduction to Laboratory Techniques" exercise. Include this graph and line
equation in the results section.
b) Calculate the moles of DCIP reduced per minute in each cuvette. To do this, use the total
volume of each reaction. Next, convert moles to molecules. Avogadro's number = 6.02 x 1023.
c) Divide molecules of DCIP by total chlorophyll (in µg) added to each cuvette. This final step
yields the rate of photoactivation in units of molecules of DCIP reduced /min/µg chlorophyll.
You must provide one sample calculation with appropriate labels/units.
5) Prepare a graph and/or table to show the effects of your independent investigation.
Discussion Section
1) Summarize all of your results in the first paragraph of your discussion.
2) Discuss your observations of the effect of light intensity on reaction rate. Was saturation
observed at high light intensity? Was the reaction rate linear at lower light intensities?
3) Discuss the findings of your independent investigation. Compare your experimental
investigation with the optimized condition (eg. tube #2 if appropriate). Explain your
observations by providing references from the text and/ or the scientific literature. Include these
citations in your references section.
References
1) Hill, R. (1939) Oxygen produced by isolated chloroplasts. Proceedings of the Royal Society of London. Series B, Biological
Sciences. 127(847). 192-210.
2) Alberts, B., et al., eds. (2008) Molecular Biology of the Cell, 5th ed. Garland Science.
3) Ohno, Y. (1997) NIST measurement services: Photometric calibrations. NIST Special publication 250-37. National Institute
of Standards and Technology, US. Department of Commerce. pp. 4-6.