![]() Enter the form of the Michaelis-Menton Equation as Ax/(B+x) where, A = Vmax x = substrate concentration and, B = Km. Click on the Define Function button in the Curve Fit window. A graph of your data will appear in the Graph Window (be sure you are not connecting your data points with a line).Ģ. You might want to add a point representing zero velocity at zero substrate concentration. Open Logger Pro and enter your initial velocity vs. Note on conducting a non-linear fit using Logger Pro:ġ. Discuss the relative merits of each treatment.Īpproximate k2 using Eq. Using the Beer-Lambert law (equation 19), and the molar absorptivity of dopachrome at the peak maximum of the band in the visible spectrum (3500 M–1 cm–1), convert each value of V into units of moles liter–1 sec–1.Īnalyze the dependence of velocity (V) on L-DOPA concentration (in units of moles per liter) using (1) a non-linear, curve fit to the Michaelis Menton Equation (see below) and, a Lineweaver-Burk plot and (2) either the Eadie-Hofstee plot (for students with family names beginning A-M) or the Hanes-Wolf plot (for students with family names beginning N-Z) plot. For each concentration of L-DOPA, calculate the mean initial velocity from your replicate runs, and use this value of V for further analysis. Outside Lab: Complete the Following Calculations and QuestionsĬreate a table or an Excel spreadsheet that summarizes the results. Save all of your data files (.txt files) and this Logger Pro file in a single folder and upload it to Sakai so that you have access to it outside of lab.ī. If not, you should do another run of that substrate concentration. You should see good agreement in these values for the replicate runs for each substrate concentration. The intercepts are estimates of the initial velocities of the reactions. Review the data with your TA to be sure you have sufficient results to continue with the analysis. Repeat the procedure with your second set of runs. Repeat this procedure for the other three linear fit lines. You can use these as a good estimate of the uncertainty in slope and intercept of your linear fit. The standard deviation of the slope and intercept will be added to the information box. There will also be an information box associated with each Linear Least-squares line where you will see values for the slope, intercept and correlation.ĭouble-click inside one of these information boxes. You will see Linear Least-Squares lines appear in the graph window. Click OK to close the Select Columns box. The four velocity columns should already be highlighted within this box if not, highlight all four. To do a Linear Least-Squares analysis of these velocity lines, click the button found in the button bar at t he top of the Logger Pro screen. Your velocity data should look something like this image shown here. time for runs A through D.Ĭlick Done to close the Graph Options box. Remove the checks from Absorbance in each of the four runs and put checks in the appropriate Velocity columns so as to show velocity vs. Select Graph Options from the Options menu. Then click Done in the New Calculated Column box. Go to the pull-down menu under Variables (Columns) and Select Absorbance. Select Derivative from the Calculus option. Leave check Add to All Similar Data Sets.Ĭlick on the button in the New Calculated Column dialog box. Also select the Data Set from the drop-down menu. Enter a name and a short name for this new column. A New Calculated Column dialog box opens. Select New Calculated Column from the Data menu. Steps 1-9 must be completed before leaving the laboratory The first step is to calculate reaction velocity from the absorbance vs. The kinetic data are analyzed using Logger Pro® software. Select Save from the File menu to save your work. \): Absorbance vs time with initial points removed
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