Thomas CaudlePractical Report 2: Photosynthesis (Set A)A1. Experiments on photosynthetic electron transport and photophosphorylation using isolated chloroplastsIn a preliminary stage, chloroplasts were isolated from pea leaves, to be used in all of the following experiments. The procedure can be found in the modules handbook, and no changes were made to this procedure.In this chloroplast assay, the photosynthesis reaction will be carried out in cuvettes, outside the leaves. As a result, an artificial electron acceptor needs to be used. 2,6-dichlorophenolindophenol (DCPIP) will act as the electron acceptor, and changes from a blue colour to colourless when it accepts electrons, and is therefore reduced. This reaction is often called the "Hill Reaction".i ) The reduction of DCPIP by chloroplastsThe procedure for i) can be found in the module handbook. In this particular experiment, there were no changes made. However, this procedure is used as the basis for the procedures of other assays in this practical, but with slight adjustments, which i will make clear as I get to them.Raw Data: Absorbance Values
My data and graph clearly show that as time progresses, the absorbance of the reaction mixture decreases. Therefore, the reaction mixture is becoming more colourless as the DCPIP accepts electrons and is reduced.There was one anomalous result which appears at 220 seconds, when the absorbance increases slightly after it has fallen to a low level. I have no explanation as to why this happened, other than that there may have been a smudge on the side of the cuvette which would have raised the absorbance. There is no way that the increase could be due to the DCPIP being oxidised and turning blue again, as the cuvette was in the light the whole time.ii ) The effects of an uncoupler of photophosphorylation on photosynthetic electron transport measured as DCPIP reductionFor this assay, an uncoupler of photophosphorylation will be added to the reaction mixture. The mixture is set up exactly as in the procedure of i) only with the addition of 10µl of 3 M ammonium chloride to act as the uncoupler.Raw Data: Absorbance values
From looking at the raw data and graph, it is clear to see that as time progresses, absorbance decreases, much like i). However, it is a much more rapid decrease in absorbance, which can only be due to the uncoupler.Question: Ammonium ions uncouple photosynthetic electron transport from ATP synthesis. How do they do this? Account for any differences you observe in the rate of electron transport in this experiment compared with the rate observed in i) above.The ammonium ion acts as a proton carrier and moves...