Hello,
Background: I am trying to find the Vmax and Km values for 0.25% trypsin and the substrate casein. During optimization, I have tried different time points (1-15 minutes) and different percent of casein. For different time points, I get virtually identical Vmax values, but different Km values. Plus, my colleague said these values shouldn't be affected by time. I use the Lineweaver-Burk plot to find Vmax and Km values.
My main question: in the Lineweaver-Burk plot, for 1/[S] is [S] the initial substrate concentration that I added to trypsin, or is [S] the remaining casein concentration after I have stopped the reaction at time 1-15 minute?
Thank you in advance
Jordan
First of all, I recommend that you use nonlinear regression to fit the Michaelis rate versus [S] plot directly, instead of using the L-B double-reciprocal plot. There are good mathematical reasons for this.
Second, you should begin by making a plot of product concentration versus time (called a progress curve) at each substrate concentration. Then, measure the initial rate of each progress curve. For the lower substrate concentrations, you may see the reaction slows down with time as the substrate gets used up. To measure the initial rate, draw a straight line starting at the origin and tangent to the initial time points, stopping when the data points start to fall below the line. The slope of this tangent is the initial rate.
Finally, make the Michaelis plot by plotting the initial rate versus the substrate concentration, and use a nonlinear regression computer program to fit the data to the Michaelis-Menten equation.
To answer your question, [S] is the initial substrate concentration. Michaelis-Menten kinetics requires that the initial rate is measured because it assumes that the substrate concentration is equal to the initial substrate concentration. In other words, no significant portion of the substrate has been consumed during the rate measurement.
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