Exercise 5.7 - Kinetic analysis

LEVEL II

Materials

• 8 mM DOPA pH 6.6
• Enzyme Extract, diluted to yield 10 micomoles of dopachrome in 3-5 minutes (Exercise 5.2)
• Spectrophotometer and Cuvettes
• Stopwatch

Procedure

1. Prepare a reaction blank in a clean cuvette to contain 2.5 ml of citrate buffer and 0.5 ml of enzyme extract. Use this blank to adjust your spectrophotometer for 100% transmittance. Remove the blank and save.

2. Add 2.5 ml of 8 mM DOPA, pH 6.6 to a clean cuvette.

3. Add 0.5 ml of appropriately diluted enzyme extract. Shake well and immediately insert the tube into the spectrophotometer. Record the absorbance or transmittance as quickly as possible. Designate this reading as time 0.

4. At 30 second intervals read and record the transmittance until a transmittance value of 10% (Absorbance = 1.0) is reached. Complete the following table:

   Time (Minutes)	Absorbance	Concentration of Dopachrome(mM)	Micromoles of Dopachrome
   0.5
   1.0
   2.0
   2.5
   3.0
   3.5
   4.0
   4.5
   5.0

5. Plot time in minutes (x-axis) versus the amount of dopachrome formed (y-axis).

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