- 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 approximately 1-inch square.
- Add 100 grams of the potato to a blender, along with 100 mL of sodium
fluoride (NaF). Homogenize for about 1 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 into
- 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 precipitate.
- Divide the ammonium sulfate-treated homogenate into chilled centrifuge
tubes and centrifuge at 1500 xg for 5 minutes at 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
- 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 experiment. 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.