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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 xC")
E 0, E 15, E 30, E 45, E 60 (abbreviation for "experimental group at xC")
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:
Tube C:
Tube D:
Tube E:
Part Seven
1. Label three clean cuvettes A,B,C
2. Prepare each tube as follows:
Tube A:
Tube B:
Tube C:
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.