Photosynthesis: Electron flow and the Hill reaction
Pictures
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Background
Robert Hill demonstrated that during photosynthesis water is the source
of electrons in the light reaction and not carbon dioxide by using an experimental
design similar to the one you will be using today. The conclusions drawn
from this experiment can be summarized in the equation below:
In vivo the electron acceptor is NADP+, however, in vitro
certain dyes such as 2,6-dichlorophenolindophenol (DCIP) can act as the
electron acceptor. An equation to describe this chemically is:
DCIP is blue when it is oxidized but is colorless when is reduced (has
accepted electrons). This change can be measured in a spectrophotometer
as a change in the absorbance at the 600 nm wavelength of light.
Two main points will be addressed in this experiment:
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Chemicals which act to inhibit different points of the photosynthetic light
reaction will be administered and their effect on the reaction rate well
be addressed.
Ammonia blocks the pathways ability to manufacture ATP, by short circuiting
the hydrogen ion gradient, but does not block the production of NADPH +
H+.
3-(3,4-dichloro-phenyl)-1,1-dimethylurea (DCMU) blocks the electron
transport chain early in the pathway stopping both ATP and NADPH synthesis.
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The chloroplasts will be exposed to different intensities of light and
the rate of electron transport will be assessed.
Experimental Protocol:
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There will be 4 people per lab group
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Two people will work up the chloroplasts while the other two will set up
the 8 reaction cuvettes described in the table on p. 57. DO
NOT ADD THE CHLOROPLASTS UNTIL THE REACTION IS RUN!!
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Turn on the spectronic 20 when you start to isolate the chloroplasts from
the spinach leaves.
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Be sure to keep everything chilled on ice, as described in the lab manual,
when working with the chloroplasts.
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All 4 group members will be involved in doing the reactions described in
part B.
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Each reaction is done in a single cuvette
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Do only one reaction at a time
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Add the chloroplasts to the reaction cuvette just prior to each reaction
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There will be 9 absorbance readings for each reaction (except for tubes
1 and 2)
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Take the spectrophotometer readings as quickly as possible
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Change the RT water in the beaker between each reaction
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Zero the spectrophotometer between each reaction
