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An Investigation of Light-Dependent Electron Transport Using Dcpip

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An Investigation of Light-dependent Electron Transport Using DCPIP

By Joshua Saprid

INTRODUCTION

The process of photosynthesis is a well-studied, and important, organic process, a phenomenon known even amongst our youngest. As such, an important aspect of photosynthesis is linear electron flow. Linear electron flow precedes the reduction of NADP+ to NADPH, which in turn powers the Calvin Cycle. Our experiment investigates this linear electron flow, i.e. electron transport from H2O, during the light-dependent reactions, using chloroplasts and DCPIP. The theory behind the addition and use of DCPIP (2,6 dichlorophenol-indophenol), a liquid solution with a distinctive dark blue hue, is an electron acceptor. When DCPIP accepts electrons, it is reduced and turns colourless – hence giving us an indication of the rate of electron transport taking place. Therefore, measuring the rate of colour loss of the DCPIP through absorbance readings gives us an indication, or more accurately, an estimation of the rate of electron transport. We hypothesize that, since we are investigating the light-dependent reactions, that the amount of light will be one of the major determining factors in the rate of DCPIP colour transparency.

METHOD

Seven spectrophotometer tubes were numbered and solutions A-D were added as per the volumes shown in Table 1. Tube 1 was capped and inverted several times. The spectrophotometer was calibrated using Tube 1, which contained chloroplasts and sucrose only, as the ‘blank’, to ensure any changes in absorbance for the other treatments could be attributed to the colour of the dye DCPIP. At time zero (mins.), absorbance was recorded for all treatments immediately after addition of DCPIP and mixing of contents.

Immediately, following the time zero reading, all tubes (1-7) were placed in larger plastic tubes; tube 2 in a light-proof (black) tube, tubes 6 and 7 in tubes covered in red and green cellophane, respectively. All tubes were then placed horizontally on ice, under lights. At fifteen minute intervals, readings of absorbance were taken for all treatments, except for the dark tube, which was kept in the light-proof tube for sixty minutes, after which its absorbance was measured.

Table 1. Experimental design for the electron transport experiment.

TREATMENT

BLANK       1

DARK       2

LIGHT       3

BOILED       4

DCMU       5

RED          6

GREEN     7

A

Chloroplast suspension (ml)

1.5

1.5

1.5

 -

1.5

1.5

1.5

B

Buffered sucrose (ml)

5.5

5.3

5.3

5.3

5.2

5.3

5.3

C

Boiled chloroplast suspension (ml)

 -

 -

 -

1.5

 -

 -

 -

D

0.01 M DCMU (ml)

 -

 -

0.1

 -

E

DCPIP (ml)          (added this last)

 -

0.20

0.20

0.20

0.20

0.20

0.20

RESULTS

The recorded change for the ‘dark’ tube (Tube 2) was only 0.006nm, indicating almost no change had taken place (Figure 1). On the contrary, Tube 3 experienced a significant change in absorbance – a 0.624nm decrease (Figure 1). The ‘boiled’ tube (Tube 4) had undergone a similar change as Tube 2, experiencing almost none (a 0.013nm decrease) (Figure 1). Tube 5 the same, only undergoing a 0.008nm decrease in absorbance (Figure 1). Tube 6 and 7, on the other hand, experienced a slight decrease in their absorbance, decreasing only 0.296nm and 0.164nm, respectively (Figure 1).

Figure 1. Graph of the different absorbance readings of the tubes. Note that the point for Tube 2 at time 15 should not have any data points, but is present due to Excel’s formatting of the graph. R2 values have been shown for each trendline.

[pic 1]

Table 2. Data obtained from spectrophotometer.

Tube # & Absorbance (nm)

Time (mins.)

2

3

4

5

6

7

0

0.941

0.941

0.827

0.943

0.926

0.896

15

 

0.853

0.962

1.057

1.012

0.963

30

0.584

0.837

0.943

0.748

0.797

45

0.452

0.787

0.926

0.687

0.778

60

0.935

0.321

0.814

0.935

0.632

0.732

DISCUSSION

The reason for the almost non-existent change in absorbance for the ‘dark’ tube (Tube 2) is that the light-dependent electron transport step of photosynthesis depends on photonic energy to drive the reaction. Since the test tube containing the chloroplasts and DCPIP was contained in a black falcon tube, that is, a light-proof falcon tube, no light had been able pass through to the chloroplasts, thus almost no electron transport had taken place. Tube 3, on the other hand, was exposed to light during the entirety of the 60min. experiment, and thus experienced the most dramatic change in the readings. The increased light exposure of Tube 3 greatly increased the rate DCPIP colour loss, indicating a greatly increased rate of electron transport. Tube 4, however, also experienced almost no change in its absorbance reading. This was due not with the DCPIP and light exposure, but due to the denaturation of proteins involved in electron transport. Heat shock, caused by the boiling, hastens the denaturation of the protein (Vierstra 1993). Denaturation of the protein renders it functionally inert, as heat damages the hydrogen and covalent bonds between its adjoining component amino acid chains (Xie 2003), thereby fundamentally altering its three-dimensional structure, its shape no longer suitable for its purpose. The factor affecting the virtually absent change in Tube 5, however, was due to the DCMU added during its preparation. DCMU (3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, acts as a very specific inhibitor – it blocks the plastoquinone (an electron carrier) binding site in photosystem II, thereby inhibiting electron flow. This, will in turn, greatly reduce the rate of DCPIP colour transparency. Tubes 6 and 7, although exposed to light, gave readings not as high as that of Tube 3. The coloured cellophane covering the two tubes was responsible for this. Photosynthesis in chloroplasts works by using the full spectrum of visible electromagnetic radiation to drive its reactions. Different wavelengths of this radiation have differing amounts of energy, and higher the frequency of the wavelength, the higher its energy. In the case of our tubes, the red light was absorbed and gave rise to electron transport, however Tube 2 was exposed to the full spectrum of visible light, therefore it had a more dramatic change compared to Tube 6 as the full spectrum has more energy than the red wavelength. Tube 7, with the green cellophane, experienced transmittance, as the green wavelength is reflected by the chlorophyll in the chloroplasts. These series of results seem to support the hypothesis, and hence conclude that light is a major determining factor in linear electron flow. However, an important aspect to note is that the results for the 15min. interval between zero and 15 had been modified. This was due to mistakenly blanking the spectrophotometer with the air, adding a factor of 0.17 to the results. We mitigated this error by subtracting 0.17 from the obtained data. Another potential source of error was the measurement of any fluid during pipetting, as the wrong scale could have been used and/or the miniscule scale was a little difficult to read. Another aspect to note is the R2 values present on Figure 1. As the R2 values obtained indicated that the linear trendlines were not suitable, different trendlines were tried. A trendline of a polynomial of order 4 fit the data exactly, resulting in an R2 value of 1. For the sake of preservation, and possible error, however, the original trendlines were used.

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