Investigating Activity Of Dehydrogenase Practical

Question 1

First step of investigating activity of dehydrogenase practical

Answer 1

Cut a few leaves (spinach works well) into pieces. Remove any tough stalks

Question 2

second step of investigating activity of dehydrogenase practical

Answer 2

Using a pestle and mortar, grind up the leaf pieces with some chilled isolation
solution (a solution of sucrose, potassium chloride and phosphate buffer at pH 7)
Filter the liquid you make into a beaker through a funnel lined with muslin cloth.

Question 3

third step of investigating activity of dehydrogenase practical

Answer 3

Transfer the liquid to centrifuge tubes and centrifuge them at high speed for 10 minutes.
This will make the chloroplasts gather at the bottom of each tube in a ‘pellet’.

Question 4

fourth step of investigating activity of dehydrogenase practical

Answer 4

Get rid of the liquid from the top of the tubes, leaving the pellets in the bottom.

Question 5

fifth step of investigating activity of dehydrogenase practical

Answer 5

Re-suspend the pellets in fresh, chilled isolation solution. This is your
chloroplast extract. Store it on ice for the rest of the experiment.

Question 6

sixth step of investigating activity of dehydrogenase practical

Answer 6

Set up a colorimeter with a red filter and zero it using a cuvette (a cuboid-shaped
vessel used in colorimeters) containing the chloroplast extract and distilled water.

Question 7

seventh step of investigating activity of dehydrogenase practical

Answer 7

Set up a test tube rack at a set distance from a bench lamp. Switch the lamp on.

Question 8

eighth step of investigating activity of dehydrogenase practical

Answer 8

Put a test tube in the rack, add a set volume of chloroplast extract to the tube and a set volume of DCPIP. Mix the contents of the tube together.

Question 9

ninth step of investigating activity of dehydrogenase practical

Answer 9

Immediately take a sample of the mixture from the tube and add it to a clean cuvette. Then place the
cuvette in your colorimeter and record the absorbance. Do this every 2 minutes for the next ten minutes.

Question 10

tenth step of investigating activity of dehydrogenase practical

Answer 10

Repeat steps 7 to 9 for each distance under investigation.

Question 11

what happens if dehydrogenase activity is taking place

Answer 11

If dehydrogenase activity is taking place, the absorbance will decrease
as the D CPIP gets reduced and loses its blue colour.

Question 12

what happens the faster absorbance decreases

Answer 12

The faster the
absorbance decreases, the faster the rate of dehydrogenase activity.
You can plot a graph of absorbance against time for each distance
from the light source. Then comp are your results to determine how
light intensity affects the rate of the dehydrogenase enzyme.