required practical 8- dehydrogenase activity in chloroplasts

what is dehydrogenase?

an enzyme found in plant chloroplasts that is crucial to the light dependent stage of photosynthesis. dehydrogenase catalyses the reaction where electrons are accepted by NADP in the light dependent stage

what happens to the action of dehydrogenase when a redox indicator dye is present? (e.g. DCPIP)

electrons are accepted by this instead.

how can the activity of dehydrogenase be investigated?

using DCPIP which turns from blue to colourless when it is reduced

give a method 

• remove stalks from leaves and blend. put into chilled isolation medium

• filter sample into beaker using muslin cloth + funnel. suspend beaker in ice bath to keep chilled

• centrifuge at high speed for 10 minutes to separate chloroplasts into pellet

• remove supernatant and add pellet to fresh isolation medium, store on ice

• set colorimeter to red. zero using a cuvette with chloroplast extract and distilled water

• place test tube in rack 30cm from light and add DCPIP. immediately take sample and add to cuvette, take sample and measure absorbance every 2 mins for 10 mins

  • repeat for different distances from light source up to 100cm- vary light intensity

what two control tubes need to be set up to carry out this practical?

1. 5cm cubed DCPIP, 1cm cubed water, 1cm cubed chloroplast suspension. covered in foil to stop light- stop photosynthesis

   1. 5cm cubed DCPIP, 1cm cubed water, 1cm cubed isolation medium. control tube to prove colour change due to chloroplast suspension, rather than isolation medium, as no colour change is seen

how can a test tube be prepared for this practical to act as a standard?

test tube 3 = standard (6cm cubed water, 1cm cubed chloroplast suspension) to identify end point of reaction, so time stopped at same point in each tube, fair test

why was there no midrib or stalk

doesnt have many chloroplasts

why was a blender used?

breaks open cells and releases chloroplasts

why is it important to keep the isolation medium cold?

blender releases heat which could denature enzymes, so keeping cold allows photosynthesis to continue

what risks are there?

DCPIP-irritant, wear eye protection. biohazard- allergies, wash hands

what graph can be plotted

graph of absorbance against time for each distance from light

what conclusions can be drawn.

As light intensity decreases, rate of photosynthesis also decreases- lowered light intensity slows rate of photosynthesis of the chlorophyll pigment, so overall rate of the light independent reaction will be slower. This means less electrons are released by the chlorophyll, hence the DCPIP accepts less electrons. This means that it will take longer to turn from blue to colourless.
When the DCPIP is blue, the absorbance is higher. The rate at which the absorbance decreases can therefore be used to determine the activity of the dehydrogenase enzyme. A higher rate of decrease, shown by a steep gradient on the graph, indicates the dehydrogenase is highly active