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Practical 8 Method

This experiment investigates the effect of ammonium hydroxide on the rate of dehydrogenase activity in chloroplasts. Spinach leaves are blended with isolation medium and filtered to produce a chloroplast suspension. Test tubes containing the dye DCPIP, with and without added ammonium hydroxide, are placed in ice and exposed to light. The chloroplast suspension is added and absorbances are measured over time using a colorimeter. Ammonium hydroxide may affect the rate of electron transfer from chloroplasts to DCPIP, indicating its effect on dehydrogenase activity.

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85 views1 page

Practical 8 Method

This experiment investigates the effect of ammonium hydroxide on the rate of dehydrogenase activity in chloroplasts. Spinach leaves are blended with isolation medium and filtered to produce a chloroplast suspension. Test tubes containing the dye DCPIP, with and without added ammonium hydroxide, are placed in ice and exposed to light. The chloroplast suspension is added and absorbances are measured over time using a colorimeter. Ammonium hydroxide may affect the rate of electron transfer from chloroplasts to DCPIP, indicating its effect on dehydrogenase activity.

Uploaded by

sarah w
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Practical 8 method:

Overview of Practical

In this example experiment, I will be investigating the effect of Ammonium Hydroxide on the rate of
dehydrogenase activity in chloroplastsPlease be aware that this may not be the practical that you
have done/will be doing, but it is an example. In the exam, the question on factors affecting
dehydrogenase activity may have a completely different experiment, but the fundamental principles
will be the same.

Equipment

cold isolation medium 50cm^3 measuring cylinder beakers: 2 large, 1 medium, 1 small 8 spinach
leaves ice muslin filter funnel blender marker pen test tubes and rack

aluminium foil cold distilled water cold ammonium hydroxide solution ruler timer lamp colorimeter
and cuvettes

Risk Assessment

Ammonium Hydroxide: irritant to eyes and in cuts. wash areas contacted immediately Wear safety
glasses

Method

Put 50cm^3 of isolation medium into the medium beaker. Tear the spinach into small pieces and,
omitting the stalk and midrib, put the pieces into the isolation medium. Fill a large beaker with
partially crushed ice and put a small beaker in so that it is surrounded by ice. Wet 3 layers of muslin
with isolation medium and place them over the top of the funnel. Put the funnel into the small
beaker. Blend the leaf-isolation medium mixture for 15 seconds and then pour a small amount
through the muslin. Gently fold and squeeze the muslin. Repeat until most of the mixture has been
filtered. Label the small beaker 'chloroplast suspension'. Label a clean test tube 'A' and add 5cm^3 of
DCPIP and 1cm^3 of distilled water to it. Wrap in aluminium foil immediately and stand in the other
large beaker after filling it with ice. Label another clean test tube 'B'. Add 5cm^3 of DCPIP, 1cm^3 of
distilled water and 1cm^3 ammonium hydroxide to it. Place it in the beaker with 'A'. Label another
clean test tube 'C'. Add 6cm^3 of distilled water to it and put it into the large beaker. Label 2 test
tubes 'X1' and 'X2' respectively. Add 5cm^3 of DCPIP and 1cm^3of water to both. Put them in the
large beaker. Label 2 test tubes 'Y1' and 'Y2' respectively. Add 5cm^3 of DCPIP and 1cm^3of
ammonium hydroxide to both. Put them in the large beaker. Place a lamp 10cm away from the large
beaker containing the test tubes and switch it on. Add 1cm^3 of chloroplast suspension to each
tube. Shake 'X1', 'X2', 'Y1' and 'Y2' thoroughly and start the timer. (If 'X1' to 'Y2' have the chloroplast
suspension added at different times, record this and account for the difference later in Step 14.)
After 5 minutes, use the colorimeter to test the absorbances of the mixture of each test tube.
Record these values. Use 'C' as the standard. Repeat Steps 10-14, this time labelling with 'X3', 'X4',
'Y1' and 'Y2'.

Application to Biology

during the Light Dependent Reaction in Photosystem I, NADP acts as an electron acceptor and is
reduced this reaction is catalysed by a dehydrogenase enzyme to track this enzyme's activity, you
can use a redox indicator dye like DCPIP which are also electron acceptors and change colour when
they are reduced (e.g. DCPIP goes from blue to colourless)

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