Required Practical 8- Rate of dehydrogenase in chloroplasts Flashcards
What is dehydrogenase?
an enzyme found in plant chloroplasts that is crucial to the light dependent stage of photosynthesis. In the light dependent stage, electrons are accepted by NADP. Dehydrogenase catalyses this reaction.
How does this experiment work?
When a redox indicator dye is present, such as DCPIP, electrons are accepted by this instead. The activity of dehydrogenase can therefore be investigated using DCPIP, which turns from blue to colourless when it is reduced.
Why are the midrib and leaf stalks discarded?
Do not contain many chloroplasts
Why was isolation medium cold?
Prevent/slow enzyme activity- prevent damage to chloroplasts
Why are leaves blended for no longer than 15 seconds?
Free chloroplasts without damaging
Why was mixture filtered through muslin cloth?
Finer separations (only allow chloroplasts to be filtered into solution) remove larger cell debris
Why was leaf suspension homogenised?
Separate chloroplasts from other organelles
Slightly damaged chloroplasts provide advantage?
DCPIP can access thylakoid membrane easier
Why does DCPIP change colour?
-blue (oxidised) to colourless (reduced)
-takes place of NADP
-accepts electrons from electron transfer chain and photolysis of water
Explain the effect of ammonium hydroxide on rate of decolorisation of DCPIP hence light dependent reaction
-accepts electrons so electrons do not pass long electron transport chain to reduce DCPIP
-slows electron transfer by inhibiting enzyme NADP dehydrogenase
-affects pH so denatures proteins in e- transport chain
Suggest why a tube containing DCPIP and leaf extract in the dark is prepared
no colour change
- proves reaction is light dependent (investigate affect of light on photosynthesis)
Part of chloroplast where transfer of electrons take place?
Thylakoid membrane
Why was leaf material grinder and blended?
To rupture cell membrane- release cell organelles (chloroplasts)
Control experiment?
-Tube containing water, isolation solution and DCPIP, NO CHLOROPLASTS
-Used to determine when other tubes had decolourised fully (when DCPIP accepted electrons and become reduced)
Variables affecting experiment?
-Light intensity- affect rate of photosynthesis (photo ionisation) - so effect rate of decolorisation
-Temperature- increase temp=increase rate photosynthesis, molecules more kinetic energy, collide more frequent
-volume DCPIP- higher= longer for decolorisation as more electrons need to be accepted
