Exercise 5.1 - Extraction of Tyrosinase

LEVEL I

Materials

• Potatoes
• Paring Knife
• Blender
• 0.1 M NaF
• Rubber Gloves
• Saturated Ammonium Sulfate (4.1 M @25° C)
• Volumetric Cylinders (50 ml, 100 ml, 250 ml)
• Cheesecloth
• Beakers (100 ml, 250 ml)
• Chilled Centrifuge Tubes (30-50 ml)
• Refrigerated Centrifuge
• 0.1 M Citrate Buffer, pH 4.8
• Glass Stirring Rod

Procedure

1. Peel a small potato and cut into pieces about 1 inch square.

2. 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.

3. Pour the homogenate (mixture) through several layers of cheesecloth and into a beaker.

4. 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.

5. Divide the ammonium sulfate-treated homogenate into chilled centrifuge tubes and centrifuge at 1,500 xg for 5 minutes @ 4° C.

6. 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.

7. 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.

8. Again divide the solution into centrifuge tubes and recentrifuge at 300 xg for 5 minutes at 4° C.

9. 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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