Exercise 5.7 - Kinetic analysis

LEVEL II

Materials

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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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu