RP 8 - Investigating Dehydrogenase Activity

• 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 such as DCPIP is present, 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.
• In this method, the named variable is light intensity.
• The method involves grinding a leaf sample using a pestle and mortar and placing it into a chilled isolation solution.
• The sample is filtered into a beaker using a muslin cloth and funnel and suspended in an ice water bath to keep it chilled.
• The sample is transferred to centrifuge tubes and centrifuged at high speed for 10 minutes to separate chloroplasts into the pellet.
• The supernatant is removed and the pellet is added to the fresh isolation medium.
• The isolation solution is stored on ice.
• The colorimeter is set to the red filter and zeroed using a cuvette containing chloroplast extract and distilled water.
• A test tube is placed in the rack 30cm from the light source and DCPIP is added.
• The absorbance of the sample is measured using the colorimeter every 2 minutes for 10 minutes.
• The 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.
• As the light intensity decreases, the rate of photosynthesis also decreases 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.
• This means that less electrons are released by the chlorophyll, hence the DCPIP accepts less electrons.
• 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.