Required Practical 8: Dehydrogenase Activity

Overview of Practical

• In this example experiment, I will be investigating the effect of Ammonium Hydroxide on the rate of dehydrogenase activity in chloroplasts
• Please 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)