NAME: Latiyah Timothy LAB PARTNER: Jasiel Mohammed

ID# 816012983 DATE: 19th November 2019

Course Code: Bioc 2069

Title of Lab: The Hill Reaction in Isolated Chloroplasts.
Aim:
 To isolate and quantify chlorophyll from plant tissues.
 To understand the effects of light intensity and inhibitors on the light reactions of
photosynthesis.
Theory:
Photosynthesis is the process by which plants and some bacteria trap light energy to which is then
used for carbohydrate synthesis and ATP synthesis. (Nelson and Cox 2017) This process can be
summarized by the following equation:
6CO2 + 12H2O→C6H12O6 + 6CO2 + 6H2O
The process of photosynthesis can be distinguished into two reactions: the light-dependent reactions
which sunlight provides energy for ATP and NADPH synthesis and the light-independent reactions by
which carbon dioxide is fixed to form triose phosphates in The Calvin cycle. These processes all
occur in the chloroplasts of photosynthetic eukaryotic cells. (Nelson and Cox 2017)
In this lab, the light reactions of photosynthesis were investigated. In the light-dependent reactions of
photosynthesis, excited electrons from the chlorophyll "special pair" are moved through a series of
electron carriers that are situated in the thylakoid membrane of chloroplasts. This energy generates the
synthesis of ATP in the stroma and the final electron acceptor is reduced to form NADPH. (Burg, et
al. 2015) This reaction can be summarized by the overall equation:
2 H2O + 2 NADP+ + 3 ADP + 3 Pi + light → 2 NADPH + 2 H+ + 3 ATP + O2

Fig 1:Chloroplast

This reaction can be explained by what is known as the Z-scheme. The Z-scheme is a representation
of the photosynthetic electron transport chain, it also shows the movement of electrons from a more
positive reduction potential (water) to a more negative reduction potential (NADPH).
Fig 2: Z-Scheme (Burg, et al. 2015)

In this lab, the Hill reaction was done as well as the effects of inhibitors and light intensity on the
reaction was also investigated. The Hill reaction was first discovered by Robin Hill in 1937 when he
demonstrated that oxygen production occurs independently of carbon fixation. Hill demonstrated that
when illuminated by sunlight and in the presence of an electron acceptor plants produce oxygen.
(Nelson and Cox 2017) To reproduce the Hill reaction in this lab, DCPIP (2,6-
dichlorophenolindophenol) was used as an artificial electron acceptor. When oxidized DCPIP is blue
and colourless when reduced. The general equation for this reaction is :
(blue) (colourless)
H2O + DCPIP DCPIP(reduced) + 1/2 O2
The Hill Reaction is used to measure the rate of photosynthesis in chloroplasts. While investigating
the rate of photosynthesis the effects of inhibitors and light intensity was also investigated. One such
inhibitor is ammonia. Ammonia acts as uncoupler in this reaction whereby phosphorylation and
electron transport is separated. However, this means just means that ATP synthesis will be inhibited
while the reduction of the electron acceptor continues.
DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a herbicide that blocks the flow of electrons as
well as phosphorylation by the blocking the plastoquinone binding site of Photosystem II. (Metz, et al.
1986) As such the electron acceptor cannot be reduced. At high concentrations of DCMU the
reduction of DCPIP is completely blocked, however at low concentrations some DCPIP can still be
reduced.

Apparatus and Materials:

1. 0.35M NaCl-0.02M Tris buffer, pH 7.5
2. 0.4mM DCIP
3. 0.01 N ammonia
4. 10-4 M DCMU
5. Purified sand
6. Fresh spinach
Procedure:
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 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 were 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 chlorophyll 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 benchtop 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.
Measurement of Caratonoid content
0.5mL of the well-mixed chloroplast suspension was pipetted in a small thick-rimmed, test tube.
4.5mL of 80% acetone in water was added. It was then centrifuged at 200rpm for 10minutes on the
benchtop centrifuge. The supernatant was transferred to a glass cuvette and the absorbance was read
at the following wavelengths (470nm, 646nm and 664nm). 80% acetone was used as the blank. The
supernatant was discarded after recording the absorbance.
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 datasheet.
Tube 2 was then immediately placed 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
datasheet. 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 datasheet.
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 were recorded in the datasheets.
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.
Results:
Table 1: The effect of inhibitors on the rate of the Hill reaction
Absorbance readings at 600nm at each time interval

TUBE
Time (minutes) 1 2 3 4
0 - 1.658 1.136 1.354
1 - 1.573 1.128 1.214
2 - 1.598 1.087 1.180
3 - 1.476 1.025 1.147
4 - 1.414 0.998 1.101
5 - 1.324 0.975 1,065
6 - 1.342 0.945 1.033
7 - 1.241 0.927 0.970
8 - 1.221 0.867 0.963
9 - 1.128 0.869 0.913
10 1.539 1.081 0.795 0.906

Table 2: The Total change of absorbance at each time interval

TUBE
Time (minutes) 2 3 4
0 0 0 0
1 0.085 0.008 0.140
2 0.160 0.049 0.174
3 0.182 0.111 0.207
4 0.244 0.138 0.253
5 0.334 0.161 0.289
6 0.316 0.191 0.321
7 0.417 0.209 0.384
8 0.437 0.269 0.391
9 0.530 0.267 0.433
10 0.577 0.341 0.448

Table 3: The Effect of Light Intensity on the rate of the Hill reaction
Absorbance readings at 600nm at each time interval
TUBE
Time (minutes) 1 2 3 4
0 - 0.879 0.712 0.516
1 - 0.709 0.613 0.464
2 - 0.627 0.520 0.416
3 - 0.532 0.483 0.395
4 - 0.432 0.433 0.285
5 - 0.361 0.402 0.277
6 - 0.301 0.346 0.231
7 - 0.272 0.261 0.152
8 - 0.225 0.228 0.146
9 - 0.172 0.221 0.130
10 0.722 0.116 0.183 0.110
Table 4: The Total change of absorbance at each time interval

TUBE
Time (minutes) 2 3 4
0 0 0 0
1 0.170 0.099 0.052
2 0.252 0.192 0.100
3 0.347 0.229 0.121
4 0.447 0.279 0.231
5 0.518 0.310 0.239
6 0.578 0.366 0.285
7 0.607 0.451 0.364
8 0.654 0.484 0.370
9 0.707 0.491 0.386
10 0.763 0.529 0.406

Table 5: Determination of Carotenoid Content.

Wavelength (nm) Absorbances

470 0.214
646 0.073
664 0.161

Calculations:
Part A: Determination of Chlorophyll Content

 C= A/(ε x l)
0.110
Absorption = 0.110 ∴ Concentration = = 0.00319mg
34.5×1

In 0.1mL, the concentration would therefore contain 0.00319mg

If 0.1mL of chloroplast suspension contained 0.00319mg of chloroplasts, then 1mL (diluted)

0.00319
would contain 0.1
× 1 =0.0319 mg/mL of chloroplasts.

0.0319
Then 10ml of diluted solution would contain 0.1
× 1 = 0.319 mg/l

∴ 1 ml of stock solution would contain 0.319 mg

 Preparing working chloroplast suspension:

No dilution was created.

 Yield per gram-wet-weight of plant tissue:

Concentration of chloroplast suspension: 0.319 mg/mL Volume

of chloroplast stock: 5.5 mL
0.319×5.5
Therefore yield per gram wet weight = 20
= 0.0877 mg/g
 Part B: Determination of Carotenoid Content:

Using the equation:

Cha = 12.25(A664) – 279(A646)
Chb = 21.5(A646) – 5.1(A664)
C x+c = (1000(A470) – 1.82Ca – 85.02Cb)/198
Where: Cha =chlorophyll A
Chb = chlorophyll B
C x+c =Carotenoids

Cha = 12.25(0.161) − 279(0.073)= -18.395

Chb = 21.5(0.073) − 5.1(0.161) =0.7484
Cx+c = (1000(0.214) − 1.82(-18.395) − 85.02(0.7484) )/ 198
= 0.928mg
Graph 1:

Graph of Effects of Inhibitors on Change of Absorbance

(600nm) of DCPIP vs Time (min)
0.7

0.6 y = 0.0549x + 0.0239

R² = 0.9854
0.5
Absorbance (600nm)

y = 0.0408x + 0.0725
0.4
R² = 0.954
0.3
y = 0.0332x - 0.0074
0.2 R² = 0.9819

0.1

0
0 2 4 6 8 10 12
-0.1
Time(min)

Tube 2 Tube 3 Tube 4

Scale : X axis : Y axis :

Graph 2:

Graph of Effects of Light Intensity on Change in Absorbance

(600nm) of DCPIP vs Time(min)
0.9
0.8 y = 0.0711x + 0.103

0.7
Absorbance (600nm)

0.6 y = 0.0511x + 0.0564

0.5
0.4
0.3 y = 0.0429x + 0.0178
0.2
0.1
0
0 2 4 6 8 10 12
Time (min)

10 cm 40 cm 60 cm

Scale : X axis : Y axis :

 Discussion:
The Hill reaction is used to measure the rate of photosynthesis, specifically the light-dependent
reactions. DCPIP is a Hill reagent that acts as the final electron acceptor in the photosynthetic electron
transport chain. (Nelson and Cox 2017)In this lab, the effects of inhibitors such as DCMU ammonia
on the photosynthetic rate as well as light intensity were investigated.
In this lab, the determined concentration of chlorophyll that was used was 0.319mg/ml. The overall
yield from 20g of spinach leaves was determined to be 0.0877mg/g. The carotenoid content was also
determined. The result showed that 0.928 mg was present in the supernatant.
The second part of the lab was the investigation of the inhibitors ammonia and DCMU of the
photosynthetic rate. Tube 1 was used as a control and gave an absorbance reading of 1.539 after 10
minutes with no illumination. Tube 2 contained only the electron acceptor and had the highest change
in absorbance. This was expected as there were no inhibitors blocking the reduction of DCPIP.
Tube 3 contained the uncoupler ammonia. From the graph and results obtained it can be seen that the
change in absorbance is steady and fairly linear but lower than that of tube two. This was expected as
ammonia only blocks ATP synthesis and not electron flow, therefore, DCPIP was still being reduced.
Tube 4 and the slowest changes in absorbance. This was expected because the herbicide, DCMU was
in this tube. DCMU blocks electron flow, however, the concentration of DCMU used was not high
enough to completely block the flow of electrons, hence DCPIP was still being reduced slowly.
(Metz, et al. 1986)
The effect of light intensity was also investigated in this experiment. Tube 1 was used as control and
was situated in the dark for 10 minutes before an absorbance reading took place. It was seen the light
intensity has some effect on the photosynthetic rate as the absorbance of tube 1 (0.722) was less than
the change absorbance in tube 2 after 10 minutes (0.763).
Tube 2 which was 10 cm away from the light source had the greatest change in absorbance of 0.763
after 10 minutes. Tube 3 was situated 40cm away from the light source and had a total change in
absorbance of 0.529 after 10 minutes. Tube 4 was situated 60cm away from the light source and had a
total change in absorbance of 0.406. These results were expected as light intensity is inversely
proportional to the square distance and as such the intensity of light decreases as distance increases.
This means that as the tube 4 had the lowest change in absorbance because the light was less intense
and DCPIP reduced slower than that of tube 2.

Sources of Error/ Precautions:

 Ensure that the DCPIP is foiled and put away when not in use as it is light sensitive.
 Ensure that the light source is directly hitting the tubes, as this can introduce error such as
inaccurate readings into the experiment.

Additional Discussion:
3. Tube 2 shows the most rapid reaction. This is expected because tube 2 has no inhibitors, therefore
there is free flow of electrons and DCPIP is reduced more quickly than that of tubes 3 and 4 which do
contain inhibitors.
4. The light reactions of photosynthesis are considered "linear" where the electrons flow from one
carrier to the other. However, in the presence of DCMU the linearity of photosynthesis is disrupted as
PSII is blocked. The appearance of the curve on the graph may be a reflection of inhibition of PSII
and fewer electrons being flowed to the electron acceptor and some being accepted by DCMU. (Metz,
et al. 1986)
5. Hills reaction may have been going before the absorbance for the 0-minute of tubes 2 and 3 were
taken. However, there is no evidence because the only observable evidence would be the change in
colour of DCPIP, the reaction was not fast enough to determine any changes in colour without the
spectrophotometer.
REFERENCES

Burg, Jeremy.M, Lubert Stryer, Gregory J Gatto Jr, and John L Tymoczko. 2015. Biochemistry. W.H
Freeman.
Metz, J, H Pakrasi, M Seibert, and C Arntzer. 1986. ""Evidence for a dual function of the herbicide-
binding D1 protein in photosystem II." FEBS letters 205.
Nelson, David, and Michael Cox. 2017. Lehninger's Principles of Biochemistry. New York: W.H
Freeman.