Exercise 5.4 - Effects of pH

LEVEL I

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

1. 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

2. 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.

3. 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

4. Plot pH (x-axis) versus Reaction Velocity (y-axis)

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