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Date: Wednesday 6th October 2010

Course Code & Title of Lab: BIOL2363- The Hill Reaction in Isolated Chloroplasts

Results:

Table 1: Absorbance at 600nm for each time interval for Experiment B

Time (min)

0 1 2 3 4 5 6 7 8 9 10

Abs. 600nm

Tube 1 0.542

Tube 2 0.790

0.634

0.607 0.590 0.561 0.486 0.435 0.326 0.304 0.301 0.205

Tube 3 0.818 0.722 0.643 0.567 0.542 0.564 0.494 0.486 0.436 0.407 0.350

Tube 4 0.812 0.746 0.685 0.596 0.593 0.579 0.492 0.472 0.458 0.413 0.388

Table 2: Absorbance at 600nm for each time interval for Experiment C

Time (min)

0 1 2 3 4 5 6 7 8 9 10

Abs. 600nm

Tube 1 0.542

Tube 2 0.841

0.772

0.670 0.605 0.594 0.510 0.433 0.404 0.370 0.334 0.293

Tube 3 0.933 0.778 0.760 0.712 0.685 0.665 0.605 0.556 0.495 0.462 0.405

Tube 4 0.884 0.822 0.794 0.769 0.699 0.643 0.570 0.546 0.378 0.132 0.112

Table 3: Total change in Absorbance (Δ A ) at each time interval for Experiment B

Time (min)

0 1 2 3 4 5 6 7 8 9 10

Abs.Δ600nm

Tube 2 0

0.156

0.183 0.200 0.229 0.304 0.355 0.464 0.486 0.489 0.585

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Tube 3 0 0.096 0.175 0.251 0.276 0.254 0.324 0.332 0.382 0.411 0.468

Tube 4 0 0.066 0.127 0.216 0.219 0.233 0.320 0.340 0.354 0.399 0.424

Table 4: Total change in Absorbance (Δ A ) at each time interval for Experiment C

Time (min)

0 1 2 3 4 5 6 7 8 9 10

Abs.Δ600nm

Tube 2 0 0.069 0.171 0.236 0.247 0.331 0.408 0.437 0.471 0.507 0.548

Tube 3 0 0.155 0.173 0.221 0.248 0.268 0.328 0.377 0.438 0.471 0.528

Tube 4 0 0.062 0.090 0.115 0.185 0.241 0.314 0.338 0.506 0.752 0.772

Calculations:

Calculating concentration of chloroplasts in the chloroplast suspension:

C= A/(ε x l)

Absorption = 0.230 ∴ Concentration = .23

34.5×1 = 6.67×10−3mg

In 10mL, the concentration would therefore contain 6. 67×10−2mg

If 0.1mL of chloroplast suspension contained 6. 67×10−2mg of chloroplasts, then 1mL would

contain 6. 67×10−1mg/mL of chloroplasts.

Preparing working chloroplast suspension:

Concentration of chloroplast suspension: 6. 67×10−1mg/mLVolume of chloroplast stock: 3.9mLNew concentration: 0.4mg/mL

C1V1=C2V2

Volume of working chloroplast = 6.67×10−1×3.90.4

= 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:

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Concentration of chloroplast suspension: 6. 67×10−1mg/mLVolume of chloroplast stock: 3.9mL

Therefore yield per gram wet weight = 6.67×10−1×3.920

= 0.1287 mg/mL

Calculating Δ Absorbance:

Δ Absorbance = Initial absorbance – absorbance @ specified time

E.g. Expt. B, Tube 2

Time (min) 0 1Abs600 Tube 2 0.790 0.634

Δ Absorbance after 1 minute = 0.790- 0.634= 0.156

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Discussion:

Photosynthesis is the process by which solar energy is trapped and used to drive the synthesis of carbohydrate from carbon dioxide and water. It occurs in green plants, algae and photosynthetic bacteria (Hames and Hooper 2005).

6CO2 + 12H2O C6H12O6 + 6O2 + 6H2O

In green plants and algae, photosynthesis takes place in the chloroplasts.

(Nelson and Cox 2008)

The Hill Reaction is the portion of the light reactions in which electrons from water are transferred to an electron acceptor thereby reducing the acceptor (Lodish, et al. 2007).In chloroplasts, the final electron acceptor is NADP+ which is reduced to form NADPH.

2 H2O + 2NADP+ + (light, chloroplasts) → 2 NADPH2 + O2

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Fig 1: Chloroplast Structure

Fig. 2: The relationship between the electron flow and electron carriers in the cell surface membrane(Taylor, Green and Stout 1997)

In the first part of the experiment, chloroplasts were extracted from spinach leaves via differential centrifugation. Differential centrifugation involves stepwise increases in the speed of centrifugation. At each step, more dense particles are separated from less dense particles, and the successive speed of centrifugation is increased until the target particle is pelleted out (Taylor, Green and Stout 1997).

In this case, the spinach leaves were homogenized and filtered into a centrifuge tube. Homogenization was necessary to rupture the cell walls and to produce a mixture of large and small molecules and membrane-bound organelles. When examined under a microscope the liquid contained grains of sand and green tissue. After the first centrifugation, the supernatant was decanted into another centrifuge tube. A sample of the supernatant was observed under the microscope and it contained green tissue. This supernatant was again centrifuged at a higher rpm and the target particle, the chloroplasts were pelleted out.

Tris-NaCl was added to the spinach cells to act as a buffer solution to prevent osmosis and rupturing of the membrane vesicles and organelles.

The chlorophyll content in the chloroplast suspension had to be determined. Chlorophyll is a photosynthetic pigment present in plants. The role of the pigment is to absorb light energy and convert it into chemical energy.

(Taylor, Green and Stout 1997)Fig. 3: Structure of chlorophyll

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Acetone in water was added to 0.1mL of chloroplast suspension before recording the absorbance. The purpose of the acetone in water was to dissolve the membrane of the chloroplast so that the chlorophyll can spill out into solution and hence its concentration determined.

In chloroplasts, the final electron acceptor is NADP+ which is reduced to form NADPH however, in this experiment, an artificial electron acceptor DCPIP was used. DCPIP changes colour from blue to colourless as it is reduced and hence the change in absorbance of this dye makes the Hill Reaction measurable.

2 H2O + 2DCPIP + (light, chloroplasts) 2DCPIPH2 + O2

(BLUE) (COLOURLESS)

(Mills 2007)Fig. 4: Reaction scheme for the reduction of DCPIP

In this experiment, spectrophotometric analysis is utilized. Spectrophotometric techniques are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer (University of Central Arkansas 2008).

When light shines on photosystems I and II, high energy electrons are released by the chlorophyll molecules in the photosystems. An electron from P680 or P700 is boosted to a higher energy level and captured by an electron acceptor (Taylor, Green and Stout 1997).

(Taylor, Green and Stout 1997)

Fig 5: Electron flow in cyclic and non cyclic photophosphorylation

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In experiment B, the effect of the inhibitors Ammonia and DCMU were investigated.

A blank was prepared using Tris-NaCl buffer to maintain chloroplasts’ integrity, distilled water and chloroplast suspension. No DCPIP was placed in this tube hence no electron acceptor was present.

Tubes 1 & 2 contained the same solutions: buffer, DCPIP, water and chloroplast suspension. However, tube 1 was wrapped in aluminium foil and its absorbance read after 10 minutes. Tube two was placed 25cm away from a light source and its absorbance read every minute for 10 minutes. The purpose of the aluminium foil in tube 1 was to prevent the entry of light into the tube since light energy is necessary for the excitation of electrons in the photosystems of the chloroplasts. The absorbance readings of tube 2 decreased gradually over the 10 minute period and hence a change in absorbance was noted as seen in Graph 1. This was due to the tube being exposed to a light source emitting light energy onto the contents in the tube. This light energy caused the photosystems in the chlorophyll molecule to release high energy electrons and instead of falling back down into the photosystem, the electrons were captured by the electron acceptor, DCPIP. The DCPIP was then reduced to DCPIPH2. Reduced DCPIP is colourless and hence resulted in the decrease in absorbance over time.

After 10 minutes, the absorbance of tube 1 was taken and it was higher than the absorbance reading of tube 2 after the 10 minute period. This was due to less reduced DCPIP present in tube 1 since there was minimum exposure to light as compared with tube 2.

Tube 3 contained buffer, DCPIP, ammonia and chloroplast suspension. The absorbance reading of tube 3 decreased gradually over the 10 minute period and a change in absorbance was present as seen in Graph 1. However, the Δ Absorbance was not as large as that of tube 2. This was due to the presence of the ammonia inhibitor in tube 3. Ammonia acts as an uncoupler of photosynthesis by passing across membranes, thereby destroying the pH gradient across the

thylakoid membrane (Smith and Raven 1979). During the light reaction, light energy is received by Photosystem II, which excites a pair of electrons to a higher energy level. These electrons travel down an electron transport chain, causing H+ to diffuse across the thylakoid membrane into the inter-thylakoid space. These H+ are then transported down their concentration gradient through an enzyme called ATP-synthase, creating ATP by phosphorylation of ADP to ATP. However, the presence of ammonia disrupts this pH/proton gradient necessary for the diffusion of the H+ ions hence the process cannot continue.Therefore the presence of ammonia led to the decrease in Δ Absorbance of tube 3 as shown on Graph 1 since less DCPIP was able to be reduced.

Tube 4 contained, buffer, DCPIP, DCMU and chloroplast suspension. The absorbance reading of tube 4 decreased gradually over the 10 minute period and a change in absorbance was present as seen in Graph 1. The Δ Absorbance was not as large as that of tube 2 but similar to that of tube 3 however it was still less than the absorbance of tube 3. This was due to the presence of the inhibitor DCMU in tube 4. DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a herbicide/ weed killer used to kill unwanted plants by inhibiting photosynthesis.

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DCMU blocks the plastiquinone-binding protein of photosystem II thus preventing electron flow. This interrupts the photosynthetic electron transport chain in photosynthesis hence blocking the ability of the plant to turn light energy into chemical energy ATP (Sengbusch 2003). Consequently, the presence of the DCMU prevented the electron flow and hence reduction of the DCPIP there in tube 4. This resulted in a decrease of Δ Absorbance.

In Experiment C, the effect of light intensity on the rate of the hill reaction was investigated.

In low light intensities, the rate of photosynthesis increases linearly with increasing light intensity. Gradually, the rate of increase falls off as other factors (e.g. carbon dioxide concentration, water and temperature) become limiting (Taylor, Green and Stout 1997).

A blank was prepared using Tris-NaCl buffer, distilled water and chloroplast suspension. No DCPIP was placed in this tube hence no electron acceptor was present.

Tubes 1, 2, 3 & 4 contained the same solutions: buffer, DCPIP, water and chloroplast suspension. However, tube 1 was wrapped in aluminium foil and its absorbance read after 10 minutes. The purpose of the aluminium foil in tube 1 was to prevent the entry of light into the tube since light energy is necessary for the excitation of electrons in the photosystems of the chloroplasts.

Tube two was placed 10cm away from a light source and its absorbance read every minute for 10 minutes. The absorbance readings of tube 2 decreased gradually over the 10 minute period and hence a change in absorbance was noted as seen in Graph 2. This was due to the tube being exposed to a light source emitting light energy onto the contents in the tube. Additionally, the tube was only 10cm away from the light source; hence the intensity of the light on the tube was high. This light energy caused the high energy electrons released from the photosystem, to reduce the DCPIP present to DCPIPH2. As a result the absorbance readings decreased.

Tube 3 was placed 40cm away from the light source and its absorbance read every minute for 10 minutes. The absorbance readings of tube 3 decreased gradually over the 10 minute period and hence a change in absorbance was noted as seen in Graph 2. However, the Δ Absorbance was less for tube 3 than that of tube 2 due to the decrease in light intensity. Tube 3 was placed 40cm away from the light source as compared to tube 2 which was placed 10cm away. The light intensity therefore, was much less on tube 3; hence less light energy would have been received by the photosystems and less electrons would have become excited. This resulted in less DCPIP being reduced and the absorbance being greater in tube 3 and Δ absorbance being smaller.

Tube 4 was placed 60cm away from the light source and its absorbance read every minute for 10 minutes. The absorbance readings of tube 3 decreased over the 10 minute period and hence a change in absorbance was noted as seen in Graph 2. However, the Δ Absorbance was less in tube 4 for the first 7 minutes as compared to tubes 2& 3 but it was significantly higher after the 7th minute. Theoretically, under lower light intensities, the rate of the hill reaction is supposed to decrease since less light energy would be absorbed by the chlorophyll and hence less electrons would become excited resulting in less DCPIP being reduced. The expected results for this experiment are plotted in Graph 3.

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The expected results show a greater Δ absorbance value for tube 2 followed by tube 3 and then tube 4.

Sources of Error:

For tube 4, experiment C, the erroneous results were due to other light sources being available. Even though the light source for our group was 60cm away from tube 4, the light sources for groups nearby were closer than 60cm from our tube 4. Therefore the tube received light energy at all sides resulting in a greater light intensity being received than the previous tubes.

Additional Discussion:

1. See Results Tables 3 & 4

2. See Graph 1

3. Experiment B investigated the effect of Inhibitors on the rate of the Hill Reaction. The reaction in tube 2 proceeded the most rapidly. Tube 2 contained buffer, DCPIP, water and chloroplast suspension. Tubes 3 & 4 contained the inhibitors ammonia and DCMU instead of water. The inhibitors present in tubes 3& 4 both hindered the hill reaction by destroying the pH gradient of the thylakoid membrane and blocked the electron flow in the electron transport chain thus preventing the reaction from occurring (see discussion). As a result in tubes 3 & 4, the amount of DCPIP reduced was significantly smaller than the amount in tube 2. The rate of reduction of DCPIP over time is directly proportional to the rate of oxygen evolution from the chloroplasts, and was used to measure photosynthesis. Since there were no inhibitors present in tube 2, the hill reaction was allowed to proceed and thus the light energy caused the photosystems in the chlorophyll molecule to release high energy electrons which were captured by the electron acceptor, DCPIP and was then reduced to DCPIPH2. The greater concentration of reduced DCPIP in tube 2 resulted in the greater Δ Absorbance.

4. Tube 4 (DCMU): There was a steady increase in Δ Absorbance until the 5th minute (however the gradient was less than that of tubes 2& 3). After the 5th minute, the gradient of the curve

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decreased but the Δ absorbance was still increasing until the 9th minute when there was again a decrease in gradient until the 10th minute when readings were no longer taken. The gradient of the curve of tube 4 was lesser than that of tube 2 due to the presence of the inhibitor DCMU. The DCMU prevented the hill reaction from proceeding hence the concentration of reduced DCPIP was much less than that of tube 2 where there were no inhibitors present.

The increase in Δ Absorbance showed that some reduced DCPIP was being made since the hill reaction would have occurred on some chloroplasts that were not in contact with any DCMU. However, as the gradient of the graph decreased, this meant that the hill reaction was decreasing and the DCMU was coming into contact and reacting with more and more chloroplasts.

5. The Hill reaction proceeded as soon as the first drop of chloroplasts suspension entered the tube containing DCPIP.This can be seen with the difference in absorption between tubes 2 & 3 at the zero minute reading.

Zero minute readings of Tubes 2&3 of Expt. B

Theoretically at zero minutes there is supposed to be no reaction taking place between anything in the tube, therefore the absorbance reading obtained for both tubes should be the same. However, absorbance readings obtained for both tubes were significantly different hence it can be concluded that the difference was due to varying amounts of reduced DCPIP in both tubes and reduced DCPIP can only be obtained due to the occurrence of the Hill Reaction.Additionally, the time taken as the chloroplasts suspension was added to when the zero minute reading was taken allowed for the hill reaction to proceed.

6. See Graph 2

7. The Hill Reaction: When light shines on photosystems I and II, high energy electrons are released by the chlorophyll molecules in the photosystems. An electron from P680 or P700 is boosted to a higher energy level and captured by an electron acceptor. In low light intensities, the rate of the hill reaction increases linearly with increasing light intensity. As the light intensity increases, more light energy/ photons become available. This light energy is absorbed by the chlorophyll molecules and transferred to electrons. These high energy electrons are responsible for reducing NADP+ to NADPH or DCPIP to reduced DCPIP. The greater the light intensity, the greater the light energy and the amount of electrons gaining energy. The larger amounts of electrons gaining energy, results in more NADPH or reduced DCPIP being formed.

See discussion for sources of error.

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Time (min) 0Abs600 Tube 2 0.790Abs600 Tube 3 0.818

References

Hames, David, and Nigel Hooper. 2005. Instant Notes Biochemistry 3/e. New York: Taylor & Francis Group,

Lodish, Harvey, Arnold Berk, Lawerence Zipersky, Paul Matsudiara, David Baltimore, and James Darnell. 2007. Molecular Cell Biology. W.H. Freeman,

Mills, Ben. Nuffield Advanced Chemistry. September 17, 2007. <http://www.chemistry-react.org/go/_10918.html> (accessed October 10, 2010).

Nelson, David L, and Michael M Cox. 2008. Lehninger Principles of Biochemistry. United States of America: W.H. Freeman and Company,

Sengbusch, Peter V. The Photosynthetic Membrane. July 31, 2003.< http://www.biologie.uni-hamburg.de/b-online/e24/24d.htm> (accessed October 15, 2010).

Smith, F A, and J A Raven. "Intercellular pH and its regulation." Plant Physiology, 1979: 289-311.

Taylor, D.J, N.P.O Green, and G.W Stout. 1997. Biological Science 1&2 3/e. United Kingdom: Cambridge University Press,

University of Central Arkansas. Spectrophotometric Analysis. 2008. <http://www2.bren.ucsb.edu/~keller/courses/esm223/Spectrometer_analysis.pdf> (accessed

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October 10, 2010).

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