AQA Biology A-Level
AQA Biology A-Level
Required Practical 8
Investigation into the effect of a named factor on the rate of
dehydrogenase activity in extracts of chloroplasts.
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Dehydrogenase?? is 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.
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.
Equipment list
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Leaf sample
Isolation solution
Ice water bath
Distilled water
DCPIP
Pestle and mortar
Test tubes
Test tube rack
Syringes
Pipettes
Lamp
Timer
Tape measure
Muslin cloth
Funnel
Beaker
Centrifuge
Centrifuge tubes
Colorimeter
Cuvettes
Method
In this method the named variable is light intensity
1. Remove stalks?? from leaf samples. Grind sample using a pestle and mortar and place
into a ?chilled isolation solution??.
2. Use a muslin cloth and funnel to filter the sample into a beaker. Suspend the beaker in
an ?ice water bath?? to keep sample chilled.
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3. Transfer to centrifuge tubes and ?centrifuge at high speed for 10 minutes??. This will
separate chloroplasts?? into the ?pellet??.
4. Remove ?supernatant?? and add pellet to the fresh isolation medium. Store isolation
solution on ice.
5. Set the colorimeter to the ?red filter??. ?Zero?? using a cuvette containing ?chloroplast extract
and ?distilled water.
6. Place test tube in the rack 30cm from light source and add? DCPIP??. Immediately take a
sample and add to cuvette. Measure the ?absorbance?? of the sample using the
colorimeter.
7. Take a sample and measure its absorbance? every 2 minutes for 10 minutes??.
8. Repeat for different distances from lamp up to 100 cm. This will vary the ?light intensity??.
NB: This experiment should be done in a? darkened room?? to make results more reliable. The
sample should not be put too close to the lamp as ?temperature?? may affect results.
Risk Assessment
Hazard
Risk
Safety Precaution
In emergency
DCPIP
Irritant to skin
and eyes; may
cause staining
Wear eye protection
Wash from skin/eyes Low
immediately using
cold water
Biohazard
Allergies; soil
bacteria;
contamination
Wash hands after
use
Seek assistance
Low
Lamps
Temporary
damage to
eyes
Do not look directly
at lamp
Wait for after image
to disappear; seek
appropriate
assistance if needed
Low
Do not touch
lamp/wires with wet
hands; keep liquids
away from
lamp/wires
Seek assistance
Low
Electrical
Liquids near
appliances electrical
appliances
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Risk
Level
Graph
¡ñ Plot a graph of ?absorbance?? against ?time ?for each distance from the light.
Conclusion
¡ñ As the light intensity decreases, the rate of photosynthesis also ?decreases??. This
is because the lowered light intensity will ?slow the rate of photoionisation?? of
the chlorophyll pigment, so the overall rate of the light dependent reaction will be
slower.
¡ñ This means that 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??.
¡ñ 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 that the? dehydrogenase is highly active??.
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