Biotechnology Methods » Enzymology
Determination of Km and Vmax
Materials
- Enzyme extract
- 8 mM L-DOPA, pH 6.6
- Spectrophotometer and cuvettes
- Stopwatch
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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 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:
- Plot the rate of DOPA conversion (v) against substrate concentration. This
is a 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-Burk 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/Km, the negative inverse of which is the
Michaelis-Menten Constant. The y-intercept is 1/Vmax and the slope equals
Km/Vmax.
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