Title: The Hill Reaction in Isolated Chloroplasts.

Aim: To isolate and quantify chlorophyll from plant tissues; to gain a better understanding of the
light reactions of photosynthesis by experimenting with factors which affect and inhibit
photosynthesis.

Theory: Most life on Earth depends on photosynthesis either directly or indirectly.

Photosynthesis makes both carbon and energy available to living organisms and produces the
oxygen in the atmosphere which is vital for all aerobic forms of life. Photosynthesis converts
carbon dioxide into glucose or other organic compounds using the energy from the sun. In plants,
photosynthesis uses carbon dioxide and water releasing oxygen as a waste product. The process
of photosynthesis can be summarized as follows:

6CO2 + 12H2O  C6H12O6 + 6CO2 + 6H2O

While this equation is superficially correct, the actual process of photosynthesis is extremely
complex and involves many separate reactions. The various reactions can be grouped into two
sets, the light-dependent ("light") reactions and the light-independent ("dark") reactions. As the
names suggest, the light-dependent reactions require the presence of a light source, while the
light-independent reactions do not use light. It is in the light-dependent reactions that the energy
of sunlight is trapped and converted to a form that can be used to drive other chemical reactions,
such as the light-independent reactions of photosynthesis (Calvin Cycle) in which carbohydrates
or other organic molecules are synthesized.

In the light reactions, excited electrons from the photosynthetic pigment chlorophyll are moved
through a series of electron carries that are embedded in the thylakoid membrane of the
chloroplasts. The energy obtained by this electron transport drives the synthesis of ATP in the
stroma and the final step in the light reactions is formative of NADPH. In the dark reactions, the
ATP and NADPH are used to convert carbon dioxide to carbohydrate.

This lab focuses on the Hill reaction which is the portion of the light reactions in which electrons
from water are transferred to an electron acceptor, reducing the acceptor.
 Figure 1. The two photosystems in photosynthesis

This reaction was first observed by Robert Hill in 1937 and it was he who demonstrated that
isolated chloroplasts can produce oxygen in the absence of carbon dioxide. This established that
the source of electrons used in the light was indeed water. In chloroplasts, the final electron
acceptor is NADP+ which is reduced to form NADPH but the Hill reaction can be studies in the
laboratory using an artificial electron acceptor. The dye 2,6-dichlorophenolindophenol (DCPIP)
is a useful acceptor because it changes colour as it is reduced from blue (oxidized form) to
colourless (reduced form). This allows the progress of the Hill reaction to be monitored in
isolated chloroplasts.

The Hill reaction can be inhibited by various chemicals that interfere at different steps of the
processes of electron transport and phosphorylation. The two inhibitors to be used here are
ammonia and DCMU, 3-(3,4 dichlorophenyl)-1,1-dimethylurea, a herbicide.

The objectives of this lab are to isolate active chloroplasts from spinach leaves; to measure the
rate of the Hill reaction and to examine the effects of two inhibitors on the rate of the reaction.

Procedure:

Materials:

1. 0.35M NaCl-0.02M Tris buffer, pH 7.5

2. 0.4M DCPIP
3. 0.01M ammonia
4. 10^-4 DCMU
5. Purified sand
6. Fresh spinach

The first part of this lab dealt with the isolation of Chloroplasts in leaves using centrifugation.
Fresh spinach leaves were first obtained and rinsed in cool tap water. The major veins were cut
from the leaves. 10g of the vein-free spinach leaves were then weighed out using an electronic
balance. The weighed leaves were then cut into small pieces using a scissors and placed in a
chilled mortar. Some purified sand was then sprinkled over the leaves. 20mL of Tris-NaCl buffer
solution was then measured out and placed on ice. 15mL of the ice-cold Tris-NaCl buffer was
then gradually added while grinding the cut up leaves for three minutes. The remaining 5mL of
buffer was then used to rinse the mortar. Two layers of cheesecloth was then used to filter the
ground leaf suspension into a chilled 15mL centrifuge tube. The excess juice in the cheesecloth
was then wrung out into the tube.

A drop of this suspension was observed under a microscope and observations were recorded.

The filtrate was then centrifuged for one minute in a chilled SORVALL centrifuge.

A drop of this suspension was again observed under the microscope and observations were
recorded.

The supernatant was then decanted into a clean, chilled 15mL centrifuge tube and centrifuged at
3300rpm for five minutes. The supernatant was then decanted carefully. The pellet was kept for
the next step.

A drop of the supernatant was then observed under the microscope and observations were
recorded.

1.5mL of ice cold Tris-NaCl buffer of pH 7.5 was then added to the pellet in the centrifuge tube.
Using a Pasteur pipette, the pellet was thoroughly resuspended. To ensure that the chloroplast
suspension was thoroughly mixed, the mouth of the tube was covered with parafilm and the tube
was then inverted several times. The above steps were repeated and the sample obtained was
combined with the first sample in one tube.

A drop of supernatant was then observed under the microscope and observations were recorded.

The centrifuge tube was then wrapped in foil and kept on ice. This was the chloroplast
suspension.

Part A of this lab dealt with the determination of the cholorphyll content of the chloroplast
suspension. 0.1mL of the well-mixed chloroplast suspension was first obtained, using a pipette,
and placed in a small, thick rimmed, test tube. 9.9mL of 80% acetone in water was added to the
tube. This mixture was then centrifuged at 2000rpm for ten minutes on the bench top centrifuge.
The supernatant was then transferred to a glass cuvette and the absorbance was read at 652nm.
80% acetone in water was then used as a blank. The supernatant was then discarded after
recording the absorbance. The concentration of chloroplasts in the chloroplasts suspension was
then calculated using the equation:

C = A/(ε x l)

The volume of the chloroplast suspension was then measured in a 10mL measuring cylinder. A
working chloroplast suspension was then prepared by diluting some of the original chloroplast
suspension, prepared in the first part of the lab, to a new concentration of 0.4mg/mL using cold
Tris-NaCl buffer solution. This diluted chloroplast suspension was used for the rest of the
experiment. The total yield per gram-wet-weight of plant tissue was determined.

Part B of this lab dealt with the effect of inhibitors on the rate of the Hill Reaction. For this part
of the lab, 5 test tubes were first obtained and labelled as “blank” and “1,” “2,” “3,” and “4.”
Each tube was prepared individually. Each was only prepared right before being experimented
on. To the “blank” test tube was added 3.5mL of Tris-NaCl buffer solution, 1.0mL of distilled
water and 0.5mL of the chloroplast suspension. The spectrophotometer was set to 600nm and
then zeroed.

Test tube 1 was wrapped in aluminium foil (this was the non-illuminated control). To test tube 1
was added 3.5mL of Tris-NaCl buffer solution, 0.5mL of 0.4mM DCPIP, 0.5mL of distilled
water and 0.5mL of chloroplast suspension. The mixture was mixed by inversion and the stop
clock was started as soon as the chloroplast suspension was added to the tube. Test tube 1 was
then set aside for 10 minutes. An absorbance reading was then taken after the 10 minute interval.

Test tube 2 was then prepared by adding 3.5mL of Tris-NaCl buffer solution, 0.5mL 0.4mM
DCPIP solution, 0.5mL distilled water and 0.5mL of Chloroplast suspension. The absorbance
was read at 600nm immediately after adding the chloroplast suspension. This was the 0 minute
absorbance. This value was entered in the data sheet.

Tube 2 was then immediately place 25cm away from the light source. The light was then turned
on the stop clock started. After 1 minute of illumination, the lamp was turned off and the tube
was removed. The absorbance was quickly measured at 600nm and the reading was entered in
the data sheet. The suspension from the cuvette was then poured back into tube 2. It was ensured
that all readings were taken quickly.

The previous step was repeated until 10 minutes had passed. That is, the absorbance readings
were taken at 1 minute intervals of illumination for ten minutes. The absorbance readings were
entered on the data sheet.

Test tube 3 was then prepared. To test tube 3 was added, 3.5mL of Tris-NaCl buffer solution,
0.5mL of 0.4mM DCPIP solution, 0.5 mL of 0.01M Ammonia and 0.5mL of Chloroplast
suspension. The procedure used for this tube was the same as that used for test tube 2.

To test tube 4 was added 3.5 Tris-NaCl buffer solution, 0.5mL of 0.4mM DCPIP solution, 0.5mL
of 0.4mM DCMU and 0.5mL of Chloroplast suspension. The procedure used for this tube was
the same as that used for test tube 2 and test tube 3. All data was recorded in the data sheets.

The final part of this lab, part C, dealt with the effect of light intensity on the rate of the Hill
reaction. 5 test tubes were first obtained and labelled. Test tube 1 was wrapped in foil so that no
light could enter. This was not required for the rest of the tubes. A blank was also prepared for
each individual reaction. To the “blank” tube was added 3.5mL of Tris-NaCl buffer solution,
1.0mL of distilled water, and 0.5mL of chloroplast suspension. The absorbance of this blank was
taken.

To test tube 1 was added 3.5mL of Tris-NaCl buffer solution, 0.5mL of 0.4mM DCPIP solution,
0.5mL of distilled water and 0.5mL of chloroplast solution. This tube was kept in the dark for 10
minutes. To test tubes 2, 3, 4 and 5 were added the same amounts of solution as for test tube 1.
The same procedure carried out for test tube 2 was carried out for these tubes. However the
distance from the light source for each tube as well as the length of time in front of the light
source was varied. The absorbance for each tube was recorded.

References:

D.J. Taylor, N.P.O. Green, G.W. Stout. “Biological Science 1.” In Biological Science 1, by N.P.O. Green,
G.W. Stout D.J. Taylor, 197 - 203. Cambridge University Press, 2001.

David L. Nelson, Michael Cox. Lehninger Prinicples of Biochemistry. New York: W. H. Freeman and
Company, 2007.