Experiment 2 - Isolation of Spinach Leaf Chloroplasts and Study of the Thylakoid Electron Transport PDF

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Isolation of Spinach Leaf Chloroplasts and Study of the Thylakoid Electron Transport Chain...

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Experiment 2: Isolation of Spinach Leaf Chloroplasts and Study of the Thylakoid Electron Transport Chain Harshita Arora 7865871

2. Photosynthesis is used by the plants to capture light energy and convert it into ATP and NADPH through a series of reactions which occur in the thylakoid membrane located in chloroplast. The interior of chloroplast is divided by this membrane into compartments called stroma and lumen1. Thylakoid membrane have complexes called photosystems embedded, which harvest photons of light. Chlorophyll molecules in the photosystems are excited by these photons, starting reduction/oxidation reactions where electron carriers carry electrons down the electron transport chain(ETC)1. The movement of electrons is also responsible for moving protons against their concentration gradient and into the lumen which will be used for ATP production. The rate of electrons flow through the ETC depends on the intensity of the light and the proton concentration gradient between stroma and lumen1. The chloroplast, which is isolated from the spinach leaves, will be used to determine how the light intensity and an uncoupler will affect the proton concentration gradient1. An artificial electron carrier (DCPIP) will be used and reduced, and its absorbance will be measured to determine the rate of the electron flow. Effect of light intensity will be determined by adding DCPIP, varying the distance of chloroplast mixture from light and measuring the absorbance difference before and after the light exposure while the uncoupler’s effect will be determined by adding various amounts of ammonium chloride to the solution of chloroplast and DCPIP, and measuring the absorbance, after and before addition, to determine the effect it has on the flow of electrons1.

3.

Table 1: Rate of Reduction for Part B Assay Distance from lamp (cm) 1 60 2 50 3 40 4 30 5 20 6 15 7 10

Change in Absorbance after 2 mins (Abs/2mins) 0.0231 0.0268 0.0324 0.0444 0.0620 0.0620 0.0712

Change in Absorbance after 1 min (Abs/min) 0.0116 0.0134 0.0162 0.0222 0.0310 0.0310 0.0356

Rate of reduction of DCPIP (mole/minute) 1.58E-09 1.83E-09 2.21E-09 3.03E-09 4.23E-09 4.23E-09 4.86E-09

Rate of reduction of DCPIP (μmole/minute) 1.58E-03 1.83E-03 2.21E-03 3.03E-03 4.23E-03 4.23E-03 4.86E-03

Table 2: Rate of Reduction for part C Assay Amount Change in Change in of Absorbance Absorbance NH4CL after 2 mins after 1 min (μL) (Abs/2mins) (Abs/min) 1 0 0.0731 0.0365 2 15 0.0944 0.0472 3 30 0.1055 0.0527 4 60 0.1304 0.0652 5 150 0.1434 0.0717 6 300 0.1452 0.0726

Rate of reduction of DCPIP (mole/minute) 4.98E-09 6.43E-09 7.19E-09 8.89E-09 9.78E-09 9.90E-09

Rate of reduction of DCPIP (μmole/minute) 4.98E-03 6.43E-03 7.19E-03 8.89E-03 9.78E-03 9.90E-03

Rate of Reduction (μmole/minute)

6.00E-03

5.00E-03

4.00E-03

3.00E-03

2.00E-03

1.00E-03

0.00E+00 0

20

40

60

80

100

120

Light Intensity

4. Figure 1. Rate of DCPIP reduction at various light intensities. Rate of DCPIP reduction was determined using a mixture of 1.800 mL of dH2O, 36.7mM TRIS (pH 7.0), 73.3 mM sorbitol, 14.7 mM KCl, 2.75 mM MgCl2 (buffer), 0.14mM of DCPIP, and 50 μL of chloroplast suspension. The absorbance was measured at 600nm, before placing it in front of the light source , and at 2 mins, after it was placed at various distances from the light source. The difference in absorbance values was taken to calculate the rate of the reduction. The cuvette length was 1cm. 5. Part B used DCPIP as an artificial electron acceptor in the thylakoid membrane so the flow of electrons could be monitored. For each assay, the distance, at which the assay was from the light source before the 2 min absorbance value was measured, was decreased gradually, which meant that the light intensity increased for each assay, gradually. In the graph, it can be seen that as the light intensity, of the amount of light reaching the assay, was increasing, the rate of the reduction of DCPIP, also increased. This suggests that the rate of reduction and the light intensity are proportional to one another. As the amount of light, which was reaching the thylakoid membrane in the mixture of the assay increased, the photosystems have more photons/area/time which are able to increase the flow of electrons. This is true because DCPIP’s rate of reduction increases with intensity suggesting that it is receiving electrons faster, meaning there are more photons which have been harvested by the photosystems to start the oxidation/reduction process. Therefore, more electrons are flowing through the thylakoid membrane as the intensity of light is increasing, as DCPIP is being reduced more. The makeup of the solution and light source is consistent in all assays tested, and only the light intensity changes.

6.

Rate of Reduction (μmole/minute)

1.20E-02

1.00E-02

8.00E-03

6.00E-03

4.00E-03

2.00E-03

0.00E+00 0

1

2

3

4

5

6

7

8

9

Concentration of NH4Cl (mM)

Figure 2. Rate of DCPIP reduction at various concentrations of NH4Cl. Rate of DCPIP reduction was determined using a mixture of 36.7mM TRIS (pH 7.0), 73.3 mM sorbitol, 14.7 mM KCl, 2.75 mM MgCl2, 0.14mM of DCPIP, 50 μL of chloroplast suspension, various volumes of dH2O and varying amounts of NH4Cl to create a 3.00mL solution. The absorbance values were measured at 600nm and the assays were placed 10 cm away from the light source where the absorbance reading was taken twice, once before placing it in front of the light and 2 minutes after removing it from the light. The difference between the absorbance values was used to calculate the Rate of reduction. Ammonium chloride is an uncoupler1 which, when added, uncouples the movement of electron and protons. Normally, the protons and electrons are coupled, so for every time plastoquinone is reduced and is then oxidized, protons are pushed into the lumen against their concentration gradient so it could be used later to form ATP2. When ammonium chloride is added, it dissociates into NH3 and H+, and NH3 can diffuse freely across the thylakoid membrane into the lumen where it binds to the protons in the lumen, forming NH4+ which changes how many free protons are around in the lumen2. Due to this, there fails to be a proton gradient between the as the protons in the lumen are being picked up NH3 which has diffused into the lumen. Since there is no proton gradient, the plastoquinone(PQ) can be reduced faster in order to try to push more electrons in the lumen as every time PQH2 is oxidized to PQ, it pushes 2 protons into the lumen for every 2 electrons passed on to the electron acceptor, which in this case is DCPIP. Also, PQH2 has to do less work when there is less of a proton gradient as it is not pushing against the gradient to push protons into the lumen. This allows PQH2 to be oxidized faster, and to pass the electrons to the electron acceptor at an increased rate. As the concentration

of ammonium chloride increases in each assay, the rate at which DCPIP is being reduced is also increasing, which suggests a faster rate of electron flow through the thylakoid membrane. The rate of reduction increases until the concentration of ammonium chloride reaches 4mM, after which it becomes stagnant and plateaus. After 4mM, the rate of reduction is the same, which means it is the rate determining step and is the maximum rate plastoquinone work at, no matter how much ammonium chloride is added to the solution and how little the proton gradient is. The rate at which PQ is reduced and oxidized has reached a maximum and therefore, the rate at which DCPIP receives electrons from PQ has also reached a maximum, explaining why the results plateau.

References (1) Nichols, E.R. (2021) Biochemistry II: Catabolism, Synthesis, and Information Pathways Laboratory Manual (2) Dean, Rob L., and Miskiewicz, Ewa (2003) Rates of Electron Transport in the Thylakoid Membranes of Isolated, Illuminated Chloroplasts Are Enhanced in the Presence of Ammonium Chloride. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 31 No.6, 410417....