LEVEL II

**Materials**

- Enzyme Extract
- 8 mM L-DOPA adjusted to pH 6.6
- Spectrophotometer and Cuvettes
- Stopwatch

**Procedure**

- Dilute the DOPA standard (8 mM) to obtain each of the
following concentrations of L-DOPA: 0.5 mM, 1 mM, 2 mM 4 mM,
and 8 mM.
- Repeat Exercise 5.7 for each of the substrate
concentrations listed, substituting the change in
concentration where appropriate.
- Plot each set of data and from the data calculate the
time required to convert 10 micromoles of DOPA to
dopachrome. Compute the velocity of enzyme reaction for each
substrate concentration. Fill in the following table:9
Substrate (DOPA)

Concentration (mM)Velocity

Micromoles/Minute1/s 1/v 0.5 2.00 1.0 1.00 2.0 0.50 4.0 0.25 8.0 0.125 - Plot the rate of DOPA conversion (v) against substrate
concentration in the appropriate place below. This is a
Michaelis-Menten plot.
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Michaelis-Menten plot

- Plot a double reciprocal of the values plotted in step 4;
that is, 1/s versus 1/v. This is a Lineweaver-Burke plot.
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Lineweaver-Burke plot

- Perform a linear regression analysis on the second plot and compute the slope and both y and x intercepts.

Note that the x intercept is -1/K, the negative inverse of which is the Michaelis-Menten Constant. The y intercept is 1/V and the slope equals Km/V.

Cell Biology Laboratory Manual

Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,

St. Peter, MN 56082 -- cellab@gac.edu