BIO211 Cell Biology

Isolation of the enzyme polyphenol oxidase and pigment formation

---------------------------------------------------------------------------------------------------------------------

INTRODUCTION
Pigments feature prominently in biology. Almost all of the reactions that take place in living
organisms are due to biological catalysts called enzymes. This week’s subject of investigation is
polyphenol oxidase (also known as tyrosinase, catechol oxidase or phenol monoxygenase), a
copper-containing enzyme, which is involved in pigment formation. As you may deduce from
the names given to this enzyme, it has a wide range of substrates but all of them are phenols or
derivatives of phenols.
While enzymes are recognized for catalyzing reactions in living things, we also exploit this
property of enzymes in other important areas. For instance, polyphenol oxidase can be used for
monitoring phenolic compounds in the environment. These compounds are used widely as
disinfectants, pesticides, insecticides etc. On the other hand this enzyme is also has clinical
importance since it can be used to monitor levels of the compound,
3, 4-dihydroxyphenylalanine (L-DOPA). This compound (L-DOPA) is used routinely in the
treatment of Parkinson's disease. In addition, this enzyme is responsible for the formation of
melanins. The melanins are implicated as important virulence factors in the yeast C. neoformans,
a causal agent for cryptococcal meningitis (Wand and Casadevall, 1994). In this week's
laboratory class, we will isolate polyphenol oxidase from mushrooms and then study its ability to
catalyse pigment formation.

MATERIALS

Extraction buffer ( 100mM NaH2PO4, 1mM Ascorbic acid pH 6)

Assay buffer (100mM NaH2PO4; pH 6.0)
Spectrophotometer (to be switched on as soon as you arrive in the lab)
Pestle and mortar
Mushroom
Ice
PROCEDURE

Isolation of polyphenoloxidase
1. Separate each mushroom into to cap tissue and stalk tissue.
2. Weigh out 3g of each type of tissue. 3 students in your group will isolate
polyphenoloxidase from cap, the other 3 strudents from stalk tissue.
3. Place the weighed sample into a mortar and add a small volume
(about 2ml) of extraction buffer provided
4. Grind the mushroom sample to a fine suspension using the buffer solution provided. It is
best to add small volumes (about 2ml) of the buffer solution at a time. Use the extraction
buffer sparingly so that you remain with about 3ml at end of preparing the extract.
5. Empty the suspension into an empty centrifuge tube kept in an ice bucket.
6. Rinse the pestle and mortar with the remaining 3ml of extraction buffer and add is to the
contents of the centrifuge tube.

1
7. All the extracts will be centrifuged at the same time; 15000 rpm for 10
minutes at 4°C. While we wait for everyone else to get their extracts
ready, place the centrifuge tube with your extract in the ice bucket
provided.
When the extracts have been centrifuged, collect the supernatant
(liquid part) into a clean sample bottle and place it on ice. Discard the debris (pellet) that
remains in the centrifuge tube.

Catalysis of pigment formation

In your group you have to measure six enzymatic activities: 3 different concentrations of the
substrate L-DOPA for two tissues type, the cap and the stalk.
1. Add the following solutions into a 3ml spectrophotometer tube:
- 0.3ml or 0.6 or 1ml of 4mM L-DOPA (Student 1, 2, 3 cap tissue and student 4, 5, 6 for
stalk tissue)
- 1.5ml of 100mM phosphate buffer pH 6
- 1.15ml or 0.85ml or 0.45ml of water
2. Start the reaction with 0.05ml of extract.
3. Mix the contents immediately and place the tube inside a spectrophotometer cell.
4. Record change in absorbance at 30-second intervals over the next 15 minutes.
Record all results.

RESULTS

Record your data in a table. How does the absorbance change with different concentrations of the
substrate L-DOPA? Which mushroom tissue contained the higher polyphenoloxidase activity?
Try to answer the following questions related to pigment formation:
- Some newborn African babies have light skin pigmentation but it gets darker with age. Explain
the likely cause of this.
- Peeled potatoes often turn brown on the surface but the browning gets slowed down when such
potatoes are immediately placed in water. Is there a scientific reason for this practice?