Exercise 5.3 - Enzyme Concentration

LEVEL I

Materials

• Enzyme Extract
• 0.1 M Citrate Buffer, pH 4.8
• 10 ml Pipette
• 8 mM DOPA
• Spectrophotometer and Cuvettes
• Ice Bath

Procedure

1. To determine the kinetic effects of the enzyme reaction, first determine an appropriate dilution of your enzyme extract. This will give a rate of reaction of 5-10 micromoles of DOPA converted per minute. Prepare a serial dilution of your enzyme extract. Place 9.0 ml of citrate buffer into each of three test-tubes. Label the tubes 1/10, 1/100 and 1/1000.

2. Pipette 1.0 ml of your enzyme extract into the first of these tubes (the one labeled 1/10) and mix by inversion.

3. Pipette 1.0 ml of the 1/10 dilution into the second tube (labeled as 1/100) and mix by inversion.

4. Pipette 1.0 ml of the 1/100 dilution into the third tube (labeled as 1/1000) and mix by inversion.

5. Place all of the dilutions in the ice bath until ready to use.

6. If not already done, turn on a spectrophotometer, adjust to 475 nm and blank with a tube containing 2.5 ml of citrate buffer and 0.5 ml of enzyme extract.

7. Add 2.5 ml of 8 mM DOPA to each of 4 cuvettes or testtubes. Note that each tube contains .0025 X .008 moles or 20 micromoles of DOPA.

8. Add 0.5 ml of undiluted enzyme extract to one of the tubes containing the 8 mM DOPA. Mix by inversion, place into the spectrophotometer and immediately begin timing the reaction. Carefully measure the time required for the conversion of 8 micromoles of DOPA. Note that since the cuvette will contain a volume of 3.0 ml, the concentration when 8 micromoles are converted will be 8/3.0 or 2.67 mM dopachrome. Use the data from the standard curve (Exercise 5.2) to determine the absorbance equal to 2.67 mM dopachrome. This absorbance value will be the end point for the reaction.

   The Absorbance equal to 3.33 mM dopachrome (from Exercise 5.2) = _______

   As the reaction takes place within the spectrophotometer, the absorbance will increase as dopachrome is formed. When the absorbance reaches the value above, note the elapsed time from the mixing of the enzyme extract with the 10 mM DOPA. Express the time as a decimal rather than minutes, seconds. The time should be between three and five minutes. If the end point is reached before three minutes, repeat Step 8, but using the next dilution of enzyme (i.e. the 1/10 after the undiluted, the 1/100 after the 1/10 and the 1/1000 after the 1/100).

   The rate of activity = ________________ micromoles/minute/0.5 ml of diluted extract. The dilution factor (inverse of dilution, 1,10,100 or 1000) is ______. The activity of the undiluted enzyme is ____________ micromoles/minute/0.5 ml or _____________ micromoles/minute/1.0 ml of extract.

9. For the enzyme dilution which reaches the end point between 3 and 5 minutes, calculate the velocity of reaction. Divide the amount of product formed (10 micromoles) by the time required to reach the end point.

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