Exercise 5.1 - Extraction of Tyrosinase
- Paring Knife
- 0.1 M NaF
- Rubber Gloves
- Saturated Ammonium Sulfate (4.1 M @25° C)
- Volumetric Cylinders (50 ml, 100 ml, 250 ml)
- Beakers (100 ml, 250 ml)
- Chilled Centrifuge Tubes (30-50 ml)
- Refrigerated Centrifuge
- 0.1 M Citrate Buffer, pH 4.8
- Glass Stirring Rod
- Peel a small potato and cut into pieces about 1 inch
- Add 100 grams of the potato to a blender, along with 100
ml of sodium fluoride (NaF). Homogenize for about one minute
at high speed.
Caution: Sodium Fluoride is a poison! Wear rubber gloves
while handling, and wipe up any spills immediately.
- Pour the homogenate (mixture) through several layers of
cheesecloth and into a beaker.
- Measure the volume of the homogenate and add an equal
volume of saturated ammonium sulfate. That is, if the fluid
volume of your homogenate is 150 ml, add 150 ml of ammonium
sulfate. This will cause a floculent white precipitate to
appear as many of the previously soluble potato proteins
become insoluble. The enzyme tyrosinase is one of these
proteins and thus will be found in the subsequent
- Divide the ammonium sulfate-treated homogenate into
chilled centrifuge tubes and centrifuge at 1,500 xg for 5
minutes @ 4° C.
- Collect the centrifuge tubes, and carefully pour off and
discard the fluid (supernatant). Save the pellets.
Combine all of the pellets into a 100 ml beaker.
- Add 60 ml. of citrate buffer, pH 4.8, to the pooled
pellet and stir the contents well. Use a glass rod to break
up the pellet. Continue to stir for 2 minutes while keeping
the solution cool.
- Again divide the solution into centrifuge tubes and
recentrifuge at 300 xg for 5 minutes at 4° C.
- Collect and save the supernatant. This is your
enzyme extract! Place it in an erlenmeyer flask, label
it as Enzyme Extract and place it in an ice bucket. The enzyme
tyrosinase is insoluble in 50% ammonium sulfate, but is
soluble in the citrate buffer. Keep this extract chilled for
the duration of the laboratory. Tyrosinase is stable for
about an hour under the conditions of this exercise. If not
used within this period, you will need to extract more
enzyme from a fresh potato.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org