Exercise 5.5 - Effects of Temperature

LEVEL I

Materials

• Enzyme Extract
• 8 mM DOPA pH 6.6
• Incubators or Water baths adjusted to 10, 15, 20, 25, 30, 35 and 40° C 6
• Spectrophotometer and Cuvettes
• Stopwatch

Procedure

1. Set up a series of test tubes each containing 2.5 ml of 8 mM DOPA buffered to a pH = 6.6. Place one tube in an ice bath or incubator adjusted to the following temperature; 10, 15, 20, 25, 30, 35 and 40° C

2. Add 0.5 ml of an appropriately diluted enzyme extract (to yield 10 micromoles dopachrome in 3-5 minutes) to each of a second series of tubes. Place one each in the corresponding temperature baths. Allow all of the tubes to temperature equilibrate for 5 minutes. Do not mix the tubes.

3. Beginning with the 10° C tube, and with the spectrophotometer adjusted to 475 nm and properly blanked, pour the enzyme (0.5 ml @ 10° C ) into the tube containing the DOPA and begin timing the reaction. Mix thoroughly. Note the time to reach the end point equivalent to the conversion of 10 micromoles of substrate.

4. Repeat Step 3 for each of the listed temperatures, complete the following and plot the data.

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