- Enzyme extract
- 8 mM of DOPA, pH 6.6
- Incubators or water baths adjusted to 10, 15, 20, 25, 30, 35 and 40°C
- Spectrophotometer and cuvettes
- Set up a series of test tubes, each containing 2.5 mL of 8 mM DOPA
buffered to a pH of 6.6. Place 1 tube in an ice bath or incubator adjusted
to the following temperature; 10, 15, 20, 25, 30, 35 and 40°C.
- 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
92 BIOTECHNOLOGY PROCEDURES AND EXPERIMENTS HANDBOOK
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.
- Beginning with the 10°C tube, and with the spectrophotometer adjusted to
475 nm and properly blanked, pour the enzyme (0.5 mL at 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.
- Repeat step 3 for each of the listed temperatures, and complete the following
and plot the data.