Bio161 Lab Report #5 On Electron Transport In Photosynthesis PDF

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Lab Report #5
Investigating Electron Transport in Photosynthesis...

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Lab Report #5 Investigating Electron Transport in Photosynthesis

Overview: Biological Significance: The importance of this experiment is to understand the flow of the transportation of electrons in photosynthesis and how DCIP and DCMU partake in the process. Photosynthesis is the ability of plants to absorb energy from light and convert it into energy for the plant. To do this, plants contain pigment molecules to absorb the energy of light. They are a green pigment called chlorophyll and are embedded in the thylakoid membranes of chloroplasts. In the presence of chloroplasts, DCIP will steal electrons from the light reaction. Specifically, the electrons are accepted by DCIP just before they reach photosystem I. Additionally DCMU prevents the transfer of electrons from photosystem II to the electron carrier, plastoquinone.

Hypothesis: Plants are not efficient at absorbing green light, but they are efficient at absorbing other colors of the spectrum, therefore the Hill Reaction would be better in the blue or red light than in the green light or in the dark.

Methods: (Describe how the biological materials and lab tools are used to answer questions) In the space below, describe the approach used to investigate the light reaction of photosynthesis. The description should summarize chloroplast isolation, and the role of DCIP.

To isolate the chloroplasts, spinach leaves were grinded into a paste, filtered, and centrifuged, twice so there would be three tubes After the tube was centrifuged for the first time, the supernatant was poured into a new tube and the original tube contained the first pellet. The supernatant was centrifuged again, and the new supernatant was poured into a new tube and the pellet from the second spin was labeled and was kept in it’s original tube. The two pellet tubes were resuspended, so there wouldn’t be any chunks in them. The tubes were then placed on separate slides, and observed under a microscope to see which sample had the cleanest source of

chloroplasts. For the DCIP to be detected, chloroplasts have to be diluted and the dilution was made by using a grinding buffer and the best chloroplast source. DCIP was used as a redox dye. DCIP is blue when the oxidized and colorless when reduced. Next, the standard reaction time was calculated by measuring absorbance after every minute of placing the tube in a plastic light box against a white light bulb. The standard reaction time is the amount of time it takes for half of the DCIP in the chloroplast sample to become reduced after the sample is exposed to light. Finally, to see the ability of different wavelengths of light to excite electrons in the chloroplasts, the DCIP solution absorbance was recorded before and after placing the tube in front of each different color light (red, yellow, green, blue, dark, and white) for the duration of the SRT.

Results: - see report booklet

Discussion/Conclusions: Part A (include your groups standard reaction time, the reasons for using standard reaction time, and possible explanation for why different groups had different standard reaction times. )

Our group’s standard reaction time was two minutes. The reason why using standard reaction time is important is because the standard reaction time is the amount of time it takes for half of the DCIP in a chloroplast sample to become reduced after the sample has been exposed to light. Different groups might have different standard reaction times because each sample of chloroplast is different due to various reasons like leaf age and health and how well the leaf was ground up.

Part B (effect of light wavelengths. Focus on known absorption spectrum of chlorophyll and compare results with predictions/hypothesis. Comment on possible problems ) :

The known range for which chlorophyll absorption spectrum is violet to blue and and yellow to red wavelengths and it peaks for the absorption being violet and orange respectively. In the absorption spectrum for chlorophyll it shows two peaks of purple and orange and a large

segment in which is has low to no absorption between blue and yellow The lab results show this where with the red light the absorbance difference was -.071 in which the negative value represent that there was more reduced DCIP. The yellow light does not correlate with the absorption spectrum as it shows more absorption of yellow light than red with an absorption of -.066. This could have happened if the red tube was left in the red light for a slightly longer time than the yellow or because of the interaction of the tube being tested and the light in the room from the fluorescent lamps and windows. The DCIP reacted as expected with the blue light since the largest peak in the chlorophyll absorption spectrum it had the most reduced DCIP with an absorbance difference of -.218. The same can be said with green light since according to the chlorophyll absorbance spectrum, since green would have the lowest to no absorption. The result of the test showed that the green light had an absorbance difference of -.034 which is lowest of the all the colored light. The white light test did not go as expected, since it would have all the visible wavelengths it would have been expected that it would have the most reduced DCIP but it did not. Instead it had the second largest oxidized DCIP with an absorbance difference of .008. This could have been due to the fact that the white lamp had a few burnt out LEDs in it while the color lamps did not. The Dark test went as expected it had the most oxidized DCIP which means that the DCIP did not have any electrons from photosystem I. This was due that it had little to no light going through solution which meant the photosynthesis did not happen with the chloroplast in the solution.

Part C (Effect of DCMU. Compare results with predictions and focus on known activity of DCMU ) :

In the lab section dealing with the DCMU, the predictions are proven by the results found it the lab. The initial absorbance of DCIP was 0.604 and the final was 0.614 which meant that the DCMU did its job by stealing the electrons away from plastoquinone before DCIP could steal the electrons away from the exchange between plastocyanin and photosystem I. This shows that DCMU disrupts the electron flow before electrons can reach DCIP.

Responses to Review Questions:

3.) Would you expect DCIP to easily cross membranes? Explain your answer, using what you know about chloroplasts structure and the Hill reaction.

The Hill reaction is the transfer of electrons to an electron acceptor with the presence of light in chloroplasts. Since the Hill reagent, DCIP, was used only within isolated chloroplasts, it will be most likely that DCIP would not easily cross membranes.

4.) How would DCMU affect formation of ATP? Of NADPH? Explain.

DCMU would very strongly affect the formation of ATP and NADPH. It would do so because DCMU blocks the passage of electrons from the primary acceptor of Photosystem II to the plastoquinone stage. This then interrupts the electron transport chain, which consequently reduces the plant’s ability to convert light energy to ATP. Since DCMU absorbs electron oxidized by water in Photosystem II, Photosystem I cannot be satisfied, which leads to the stop  of photosynthesis by blocking the reduction of NADP+ to NADPH....