* Potato extract will be prepared by following the directions in Part Two - Extraction of the Enzyme Polyphenoloxidase from a Potato on page 15 of the DVC BIOSC 130 Laboratory Manual.

Part Five

1. Prepare 10 test tubes. Label for each temperature tested, five in a control set and five in an experimental set: C 0, C 15, C 30, C 45, and C 60 (abbreviation for "control group at xºC")E 0, E 15, E 30, E 45, E 60 (abbreviation for "experimental group at xºC")

2. Add 2.0 mL catechol-buffer solution at pH 6 to all 10 test tubes.

3. Add 3.5 mL of water to the five test tubes of the control group.

4. Add 3.0 mL of water to the five test tubes of the experimental group.

5. When all test tubes are ready, start the reaction (Time = 0 min.) by adding 0.5 mL enzyme extract to the five test tubes in the experimental group only after they have reached the appropriate temperature. To achieve this, let test tube sit in water bath until temperature is matched, then add enzyme. No spectrophotometric readings are needed at Time = 0 min.

6. Let the reaction proceed for 15 minutes, until the endpoint is reached. Aerate periodically. Read all experimental tubes using the appropriate control test tube to calibrate the spectrophotometer to 100% transmittance. 

Part Six

1. Label 5 clean cuvettes A, B, C, D, and E. Prepare each tube as follows:

Tube A: 2.0 mL catechol-buffer solution* (This will be used to set the spec 20 at 100% transmittance.)

3.5 mL water

Tube B: 2.0 mL catechol-buffer solution3.25 water0.25 mL enzyme extract

Tube C: 2.0 mL catechol-buffer solution3.0 mL water0.5 mL enzyme extract

Tube D: 2.0 mL catechol-buffer solution2.75 mL water0.75 mL enzyme extract

Tube E: 2.0 mL catechol-buffer solution2.5 mL water

1.0 mL enzyme extract

* catechol-buffer (already made by staff): 1 part 0.006 M catechol solution1 part 0.1 M sodium phosphate buffer at pH 6

2. Shake the tubes immediately after adding enzyme. As the reaction begins as soon as the enzyme and substrate are put together, fill each test tube first with water and the catechol-buffer solution. Then measure out the appropriate amounts of the enzyme to add in test tubes B, C, D, and E in separate test tubes. Pour each of the four volumes of the enzyme into their respective test tubes in about the same time so each test starts at around the same point in time.

3. Shake each tube every few minutes to aerate to add oxygen to the solution.

4. At time intervals of 5 minutes, measure the percentage transmittance of the tube's contents with a spectrophotometer. Use Tube A to calibrate the machine at 100 % transmittance. Take readings for about 40 minutes.

5. In this experiment the tubes are all kept at room temperature throughout the experiment.

Part Seven

1. Label three clean cuvettes A,B,C

2. Prepare each tube as follows:

Tube A: 2.0ml catechol-buffer solution3.5ml water

Tube B: 2.0ml catechol-buffer solution3.0ml water0.5ml enzyme extract

Tube C: 2.0ml catechol-buffer solution2.5ml water0.5ml substance X0.5ml enzyme extract

Shake the tubes immediately and read for TIME=0. Substance X will be added before the enzyme extract, which is added last to cuvettes B & C.

3. Shake the tubes every few minutes.

4. Record the % transmittance using a Spectrophotometer at time intervals of 5 minutes for 30 mintues.

5. Use test tube A to set the machine to 100% transmittance before testing cuvettes B & C.