Enzyme Action: Experiment with Tyrosinase
Background
The purpose of this laboratory is to introduce students to the quantitative measurement of
enzyme activity by using the, enzyme, tyrosinase from a potato. Tyrosinase is found in
many plants and animals where it is responsible for a biochemical pathway beginning with
the amino acid, tyrosine, (hence the name “tyrosinase”) and leading ultimately to the
formation of melanin, a black pigment. Activity of this enzyme in part accounts for the
darkening of skin in response to sunlight and the darkening of a freshly peeled potato (or
banana, apple, or pear) when exposed to air.
The specificity of tyrosinase for the substrate tyrosine is not great. It will, in fact, react
with a variety of ring structures including its natural substrate, tyrosine, with one hydroxyl
group, and catechol, a phenol with two hydroxyl groups.
In the experiments below catechol is used as an artificial substrate for tyrosinase. In the
presence of this enzyme catechol is oxidized to ortho-quinone, which immediately
undergoes secondary reactions leading to the formation of strongly colored products. The
formation of these products is very rapid and their measurement over time indicates the
rate of enzyme activity.
Question
What is the rate of product formation during the first ten minutes of a reaction between the
enzyme tyrosinase and the substrate catechol? (Assume that both the enzyme and
substrate are present in high concentrations.)
Materials
Test tubes
Spectrophotometer 20
Potato enzyme (tyrosinase)
Catechol (0.006 M)
Phosphate buffer (0.1 M)
Distilled water
Procedure
A. Tyrosinase: was prepared from potato immediately before the laboratory by the
following procedure:
1. A medium sized (150-200 grams) potato was peeled, washed, chopped into small
pieces and homogenized in a blender for two minutes with 100 ml ice cold distilled
water. The homogenate was filtered through two layers of cheesecloth into a 400
ml beaker.
2. The filtrate was allowed to settle in a refrigerator for 10 minutes.
3. The filtrate was then poured off into a flask making sure not to disturb any of the
filtrate at the bottom of the beaker.
4. The filtrate was diluted 1:10 with ice cold distilled water and placed back in the
refrigerator until use.
5. Throughout your experimentation the enzyme should be kept ice cold.
B. The buffered substrate is prepared in a small flask according to the following
calculations.
5 ml
5 ml
15 ml
catechol (0.006 M)
sodium phosphate buffer (0.1M, pH 6.0)
distilled water (room temperature)
C. Experimental Methods for Basic Reaction
1. Turn on the spectrophotometer and let it equilibrate for 15 minutes.
2. Set the wavelength to 540nm.
3. With the lid closed, set the dark current to 0% Transmittance (T).
4. Use a kimwipe to clean any fingerprints or grease off a small test tube.
5. Use a blank tube with 4 ml of phosphate buffer placed in the spectrophotometer
and set the current to 100% T.
6. Empty the phosphate buffer back into a vial – this can be reused.
7. Set up control tubes using the following directions and place in your test tube rack
for observation:
Control Tubes: In order to determine whether the experimental solution is turning dark
because of the reaction with catechol it is necessary to set up two control tubes.
Control tube a, should contain 2 ml enzyme solution and 2 ml buffer. The
enzyme solution also contains the natural substrate tyrosine and so colored
products will form over the course of the lab.
Control tube b, should contain 2 ml buffered substrate (containing catechol) and
2 ml distilled water. This control determines whether the substrate catechol is
oxidized non-enzymatically at a perceptible rate. Read both control tubes at 0
and 10 minutes for every experiment.
8. Pipette 2 mL buffered substrate into a clean test tube and add 2 ml of enzyme
solution to the tube to start the reaction.
9. Mix the reaction tube by placing parafilm over the tubes and inverting to mix.
10. Wipe the tube clean of fingerprints.
11. Place the tube in the spectrophotometer and read the %T.
12. Record %T in notebook and convert to Absorbance (O.D.) using supplied table.
13. Take readings every 30 seconds for 10 minutes.
14. Repeat steps 8 – 13 using a clean test tube and fresh substrate and enzyme.