Exercise 5.3 - Enzyme Concentration
- Enzyme Extract
- 0.1 M Citrate Buffer, pH 4.8
- 10 ml Pipette
- 8 mM DOPA
- Spectrophotometer and Cuvettes
- Ice Bath
- 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.
- Pipette 1.0 ml of your enzyme extract into the first of
these tubes (the one labeled 1/10) and mix by inversion.
- Pipette 1.0 ml of the 1/10 dilution into the second tube
(labeled as 1/100) and mix by inversion.
- Pipette 1.0 ml of the 1/100 dilution into the third tube
(labeled as 1/1000) and mix by inversion.
- Place all of the dilutions in the ice bath until ready
- 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.
- 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.
- 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.
- 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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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org