Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College
http://homepages.gac.edu/~cellab/chpts/chpt5/ex5-4.html
Materials
- 8 mM DOPA in citrate buffer adjusted to
pH values of 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, 7.8
- Enzyme Extract
- Spectrophotometer and Cuvettes
- Stopwatch
Procedure
- Set up a series of test tubes each containing 2.5 ml of 8 mM DOPA, but adjusted to the following pH values: 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, and 7.8
- Begin with the tube containing DOPA @ pH 3.6, add 0.5 ml of the diluted enzyme extract which will convert 10 micromoles of DOPA in 3-5 minutes as determined in Exercise 5.3. Start timing the reaction, mix by inversion and insert into the spectrophotometer. Note the time for conversion of 10 micromoles of DOPA.
- Repeat Step 2 for each of the indicated pH values. Complete the following table:
pH
|
Time(Minutes)
|
Micromoles of
Dopachrome |
Velocity
(Micromoles/Minute) |
3.6
|
|
10
|
|
4.2
|
|
10
|
|
4.8
|
|
10
|
|
5.4
|
|
10
|
|
6.0
|
|
10
|
|
6.6
|
|
10
|
|
7.2
|
|
10
|
|
7.8
|
|
10
|
|
- Plot pH (x-axis) versus Reaction Velocity (y-axis)
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