Title: The Hill Reaction in Isolated Chloroplasts.

Results:

Table 1

EXPERIMENT B – The effect of inhibitors on the rate of the Hill reaction

Absorbance readings at 600nm at each time interval

Time/mins
Tube # 0 1 2 3 4 5 6 7 9 10
1 - - - - - - - - - -
2 0.930 0.823 0.803 0.786 0.709 0.629 0.584 0.464 0.438 0.429
3 0.415 0.383 0.263 0.285 0.244 0.132 0.092 0.070 0.020 0
4 0.600 0.564 0.525 0.486 0.479 0.398 0.317 0.256 0.253 0.176

Table 2

EXPERIMENT B-The change in absorbance of each tube after 1 minute intervals of

exposure to light.

Tube # 1 2 3 4 5 6 7 8 9 10
2 0 0.107 0.128 0.144 0.221 0.301 0.346 0.396 0.466 0.501
3 0 0.032 0.152 0.130 0.171 0.283 0.323 0.345 0.395 0.415
4 0 0.036 0.075 0.114 0.121 0.202 0.223 0.283 0.344 0.347

Table 3

EXPERIMENT C – The effect light intensity on the rate of the Hill reaction

Absorbance readings at 600nm at each time interval

Time (min)
Tube 0 1 2 3 4 5 6 7 8 9 10
1 - - - - - - - - - - -
2 0.930 0.823 0.803 0.786 0.709 0.629 0.584 0.464 0.438 0.429 0.930
3 0.723 0.698 0.610 0.544 0.522 0.403 0.366 0.288 0.265 0.233 0.188
4 0.307 0.302 0.255 0.288 0.191 0.121 0.097 0 0 0 0

Table 4
EXPERIMENT C-The change in absorbance of each tube after 1 minute intervals of
exposure to light.

Time (min)
Tube # 0 1 2 3 4 5 6 7 8 9 10
2 0 0.107 0.128 0.144 0.221 0.301 0.346 0.396 0.466 0.492 0.501
3 0 0.025 0.113 0.179 0.201 0.320 0.387 0.435 0.458 0.490 0.535
4 0 0.005 0.052 0.078 0.116 0.186 0.21 0.299 0.307 0.307 0.307

Calculations:

 Calculating concentration of chloroplasts in the chloroplast suspension:

C= A/(ε x l)

Absorption = 0.230 ∴ Concentration = = 6.67 mg

In 10mL, the concentration would therefore contain 6. 67 mg

If 0.1mL of chloroplast suspension contained 6. 67 mg of chloroplasts, then 1mL would

contain 6. 67 mg/mL of chloroplasts.

 Preparing working chloroplast suspension:

Concentration of chloroplast suspension: 6. 67 mg/mL

Volume of chloroplast stock: 3.9mL
New concentration: 0.4mg/mL

C1V1=C2V2

Volume of working chloroplast = = 6.5mL

Since initial chloroplast suspension is 3.9mL, (6.5-3.9)mL of Tris-NaCl buffer will be needed =
2.6mL of Tris-NaCl buffer used to dilute stock to working concentration

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

Concentration of chloroplast suspension: 6. 67 mg/mL

Volume of chloroplast stock: 3.9mL

Therefore yield per gram wet weight = = 0.1287 mg/mL

 Calculating Δ Absorbance:
Δ Absorbance = Initial absorbance – absorbance @ specified time

E.g. Expt. B, Tube 2 at 1 minute

Δ Absorbance = 0.930 – 0.823 =0.107

Discussion: The purpose of this lab was to examine the effects of the inhibitors
(Dichlorophenyl)dimethylurea (DCMU) and ammonia on the rate of the Hill reaction in isolated
Chloroplasts and also to explore the effects of different light intensities on the rate of the hill
reaction.

The Hill reaction is the portion of the light reactions in photosynthesis in which electrons from
water are transferred to an electron acceptor, reducing the acceptor.

Figure 1. The Z-scheme of electron flow in the light dependent reactions of photosynthesis (1)

In chloroplasts, the final electron acceptor is NADP+ which is reduced to form NADPH.

The Hill reaction can be studied in the laboratory by using an artificial electron acceptor which
changes colour upon reduction. The dye 2,6-dichlorophenolindophendol (DCPIP) is a useful
acceptor because it changes colour as it is reduced, from blue (when it is in its oxidized form) to
colourless (when it is in its reduced form). As the Hill reaction proceeds, the reduction of DCPIP
can easily be measured as a change in absorbance at 600 nm using a spectrophotometer.
 Blue Colourless

Chloroplast ferredoxin is involved in both cyclic and non cyclic photophosphorylation reactions
in photosynthesis. In non cyclic photophosphorylation, ferredoxin is the last electron acceptor
and reduces the enzyme NADP+ reductase. But DCPIP has a higher affinity for electrons than
ferredoxin. Thus DCPIP can be substituted for NADPH as the final electron carrier in the light
reactions. The light reactions, as was mentioned above, will reduce the DCPIP and turn it
colourless.

The inhibitor DCMU is a herbicide. It is a very specific and sensitive inhibitor of photosynthesis.
It blocks the plastoquinone binding site of the PSII, disallowing the electron flow from where it
is generated in PSII to plastoquinone. This interrupts the photosynthetic electron transport chain
in photosynthesis and thus blocks the ability of the plant to turn the light energy into chemical
energy. Since DCMU absorbs electrons obtained from the oxidation of water in the PSII, the
electron hole of PSI cannot be filled, effectively shutting down photosynthesis by blocking the
reduction of NADP+ to NADPH. It also means that the electrons which would have been
preferentially taken up by the DCPIP are taken up instead by DCMU. The DCPIP which is added
to the test tubes being examined is thus not reduced. Thus in the test tube containing DCMU and
DCPIP, a no change in absorbance should be observed, or the change should not be as great as
for a tube containing DCPIP and no DCMU.

Ammonia, acts as a metabolic uncoupler; it uncouples or separates the process of electron

transport from photophosphorylation (ATP synthesis in the presence of light). This uncoupling
agent abolishes the proton gradient necessary for photophosphorylation. Although electron
transport still functions, no ATP is synthesized. This causes an “uncoupling” of the two
processes. Since the rate-limiting step is the synthesis of ATP, the result of this uncoupling is an
increased electron flow, which should be reflected in the results.

For experiment B, Test tube 1 was prepared to serve the purpose of a blank. It contained a
chloroplast suspension and DCPIP as its main components and no inhibitors. It was also always
kept in the dark. The absorbance of this tube was taken to be used as the blank reading for all the
other test tubes.

Test tube 2 was prepared to serve the purpose of a control. It contained chloroplast suspension
and DCPIP but contained no inhibitors. When the graphs produced from the tubes containing
inhibitors were compared with the graph for this tube, the true effect of the inhibitors would be
seen. From the graph for this tube it can be seen that the change in absorbance for this tube
increased with time as would be expected. For every additional minute that the tube was exposed
to light, the DCPIP became increasing reduced and hence more colourless and the transmittance
of light through the tube consequently increased.

Test tube 3 was prepared to examine the effect of the inhibitor ammonia on the rate of the Hill
reaction. From the graph it can be seen that the change in absorbance with time, though
increasing was not as great as the change in absorbance observed for tube 2. This can be
accounted for from the fact that ammonia was inhibiting the reaction.

Test tube 4 was prepared to examine the effect of the inhibitor DCMU on the rate of the Hill
reaction. From the graph it can be seen that the rate of reduction of DCPIP was slightly lower
than that for tube 2. The lower rate can be accounted for from the fact that DCMU was being
preferentially reduced instead of DCPIP. DCPIP consequently did not become as quickly
reduced as it did in tube 2 and the transmittance of light through the tube was not as great as in
tube 2.

For both test tubes 2 and 3, the change in absorbance observed was perhaps greater than it should
have been. This could have happened because of experimental error. The 0 minute absorbance
may not have immediately been taken after adding the chloroplast suspension so some of the
DCPIP could have been reduced before the absorbance could be taken. Absorbance readings may
also not have been taken at exact one minute intervals i.e. the reaction time between readings
was slow causing the trend in results to be slightly off. Incorrect pipetting could have also taken
place with more chloroplast suspension being added than it should or less DCPIP being added
than it should.

The purpose of Experiment C was to determine the effect of light intensity on the rate of the Hill
reaction. This was done by measuring the absorbance of three different test tubes at 10cm, 40cm
and 60cm from the light source at 1 minute intervals for 10 minutes. Test tube 2 was placed
10cm from the light source. The graph produced from the changes in absorbance with time
showed a steady increase in transmittance with time. This was because with each minute that the
tube containing the chloroplast/DCPIP suspension mixture was placed in front the light source
more water molecules were split because of the PSII absorbing more light energy, more electrons
were released and more molecules of DCPIP were preferentially reduced instead of NADP. The
more molecules of DCPIP became reduced, the more the solution became colourless and the
smaller were the absorbances produced. The change in absorbance therefore became greater
producing the positively sloping curve.

Test tube 3 was placed 40cm from the light source. The graph produced from the changes in
absorbance with time showed a steady increase in transmittance with time. This was gain due to
more and more water molecules being split because of the photosystem absorbing light energy
and DCPIP taking up the electrons because of its higher affinity for the electrons than NADP.
The increase, however, was not as great as that for tube 2. Because of the increased distance
from the light source, the light rays became more diffused and many were diverging away from
leaving fewer rays to hit the tube directly. Consequently, less energy was absorbed by the
chloroplast suspension and the changes in absorbance would not be as great as that for tube 2
since less molecules of DCPIP would be reduced.
The graph produced from the changes of absorbance of tube 3 was not very different from tube
2. This was probably due to error and not taking readings fast enough in between one minute
intervals causing the changes in absorbance to be different from what they actually were. An
alternative explanation could be that at 40cm, this was the optimum light intensity at which the
chloroplasts worked to absorb and then convert the energy to chemical energy.

Test tube 4 was placed 60cm from the light source. The graph produced from the changes in
absorbance with time showed a steady increase in change in absorbance with time. However, for
this graph the increase was not as great as for tubes 2 and 3. The much greater distance from the
light source meant that many of the light rays were diverging away from the tube containing the
chloroplast suspension. Only a few rays would hit the tube directly so much less energy would
be absorbed by the photosystems and less water molecules would be split which would lead to
fewer molecules of DCPIP being reduced and the changes in absorbance not being as great as
they were for tubes 2 and 3.

A possible error that could have occurred was not taking readings at exactly one minute apart.
This would lead to the changes in absorbance per minute being incorrect, thus affecting the shape
of the curve.

For this lab, it was noted that DCPIP will revert to its oxidized state (blue) as soon as the
chloroplast suspension is removed from the light path. It was therefore important to take all
absorbance readings as quickly as possible. A precaution was also taken to do the experiment in
a semi dark room to limit the effects of ambient light. Another precaution was taken to take all
readings as quickly as possible for greatest accuracy. Chloroplast suspension was also only
added just before placing the tube before the light source. This was done so that the chloroplasts’
action on the DCPIP would be due to the light source provided and not any other ambient light in
the room.

Additional Discussion:

3) In which tube in Experiment B did the reaction proceed most rapidly? Explain.

The reaction proceeded most rapidly in tube 2, the tube without inhibitors. This was seen in the
graph produced for tube 2. The change in absorbance with time was seen to be greater in test
tube 2 than in tubes 3 and 4. This could be explained by the fact that the rate of the Hill reaction
was being measured by the rate at which DCPIP was being reduced (and hence undergoing a
colour change) and uninhibited, the greatest observable rate of reaction would be produced. In
the tubes containing DCMU and ammonia, the electrons were being prevented from being taken
up by the DCPIP because of the inhibitors which lowered the number of molecules of DCPIP
reduced with time and hence the changes in absorbance were not as great in these tubes as they
were in tube 2.
4) Explain the appearance of the curve for the tube with the DCMU (tube 4).

The curve produced by the tube with DCMU was similar in shape to the curve produced from the
changes of absorbance with time of tube 2 but the rate of reaction was seen to be not as great as
for tube 2.

The curve produced by tube 4 would be similar in shape since water molecules were still being
split in this tube at the same rate. This part of the reaction was not inhibited by DCMU. It was
more of a question of which molecule, DCPIP or DCMU would be accepting the electrons
emitted. Since molecules of DCMU were present, some of the electrons emitted would be
accepted by these molecules instead of DCPIP. Less molecules of DCPIP would thus be reduced
and consequently, fewer molecules would undergo a colour change. The change in absorbance
with time would therefore be not as great as that for a tube containing no inhibitors. Hence the
maximum rate seen on the curve would be lower.

5) Is there any evidence that the Hill reaction proceeded in tubes 2 and 3 prior to time that the 0-
min reading was taken? Explain.

Although the Hill reaction may have been taking place in the tubes 2 and 3 prior to the time that
the 0-min reading was taken due to stray light in the lab etc., there is no evidence of this. The
only observable evidence is the change in colour of DCPIP due to its being reduced and in the 0-
min reading, no DCPIP has been added yet. The reading taken at 0 minutes was the blank
reading The reference blank contains everything that is found in the sample solutions being
analyzed except for the compound of interest which absorbs light and eliminates error. Thus by
zeroing the machine, any measured absorbance is due to the presence of the solute of interest.
The presence of the Hill reaction was indicated by the change in absorbance of DCPIP. There is
no other evidence of it.

7) Discuss the relationship between the Hill reaction and light intensity. Include in your
discussion any possible sources of error in Experiment 2.

References:

David Hames, Nigel Hooper. Instant Notes in Biochemistry. New York: Taylor and Francis Group, 2005.

David L. Nelson, Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and
Company, 2008.