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Enzyme Kinetics Pre-Lab Exercise

This document provides instructions for students to analyze enzyme kinetic data for magnesium chelatase using GraphPad Prism. It describes how to: 1. Open the excel data file and copy it into a Prism data table. 2. Fit the data to the Michaelis-Menten equation to determine Km and Vmax values. 3. Transform the data to Lineweaver-Burk, Hanes-Woolf, and Eadie-Hofstee plots and calculate Km and Vmax from the linear regressions. 4. Create a single page layout in Prism with the four graphs for submission as the pre-lab exercise.

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shaheen
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Download as PDF, TXT or read online on Scribd
81 views

Enzyme Kinetics Pre-Lab Exercise

This document provides instructions for students to analyze enzyme kinetic data for magnesium chelatase using GraphPad Prism. It describes how to: 1. Open the excel data file and copy it into a Prism data table. 2. Fit the data to the Michaelis-Menten equation to determine Km and Vmax values. 3. Transform the data to Lineweaver-Burk, Hanes-Woolf, and Eadie-Hofstee plots and calculate Km and Vmax from the linear regressions. 4. Create a single page layout in Prism with the four graphs for submission as the pre-lab exercise.

Uploaded by

shaheen
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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BMOL3201 Pre-Lab Exercise for using Prism 8

Plotting XY enzyme kinetic data from an excel spreadsheet of replicates

1. Open the excel spreadsheet called “Mg-chelatase data” on iLearn:


This data is triplicate measurements of enzyme kinetic data for the enzyme magnesium
chelatase which converts protoporphyrin IX to magnesium protoporphyrin IX. The substrate
concentration for protoporphyrin IX are the “x” values.
The “x” values in the first column are concentration in µM of protoporphyrin IX.
The “y” values in triplicate are fluorescent units per min (FU/min). Note that they could be
some other measurement per minute e.g. Absorbance units per min (Abs/min).

2. Open GraphPad Prism version 8. You will have been sent an email invitation to
download and install this software. The following screen will appear.

Create an XY data table with 3 replicates. NOTE: you can create a data table with more than
3 replicates as it DOESN’T matter if there is missing data in the table.

Copy and paste the XY data into the Prism Data Table as below.
Enter the labels manually for X and Group A as above.
NOTE THE Results and Graphs and Layout on the left will appear like this once you have
completed the Pre-Lab! They will be empty now apart from a single Data 1 Table and Data 1
Graph!

3. We will now analyse the data as enzyme kinetic data.


Click on “Analyze” or the middle curve under “Analysis” which says “Curve fit using non-
linear regression” when you mouse over this button.
If you clicked on “Analyze” choose “Non-linear regression (curve fit)”.
Choose “Enzyme Kinetics – Velocity as a function of substrate” and “Michaelis Menton” as
below.
Click on the “Confidence” Tab and select “Show SE of parameters” and click “Make these
choices default for future fits.”
Then click OK.

4. This will generate a Graph called Data 1 and a Results called Nonlin fit of Data 1
If you rename the Data 1 Table to Mg-Chelatase the Results and Graph are automatically
renamed as below.
5. If you click on the Mg-Chelatase graph you will see the Graph with error bars. Note
that you can resize the graph and edit and relabel it. Right Click on the Graph and
right click will allow editing of axes and symbols etc.
a. You can also add a text box for adding the Km and Vmax from the results of
the Nonlin fit onto the graph. Make sure the units are correct.

Transforming the data table to draw Lineweaver Burke and Eddie Hofstee and Hanes Wolf
plots.
Lineweaver Burke Plot.
1. Select the Mg-Chelatase data table so you can see the data and click “Analyze”.
2. Choose “Transform” and click OK
3. Using the pull down menu at the top select Pharmacology and biochemistry
transforms.
4. Click Lineweaver-Burk (double reciprocal)
a. NOTE for Replicates click “Transform individual Y values”
5. Click OK.
6. Analyze the the Resulting Lineweaver Burke of Mg-Chelatase Data as a Linear
regression.
a. Note choose start X value at -1. So the line goes through the X-axis.

Hanes Woolf Plot and Eadie Hofstee plots.


1. Repeat the procedure for Lineweaver Burke Plot but this time choose Hanes Woolf.
a. Note that for this Hanes Woolf plot the start X value for the linear regression
should be minus 0.4.
2. Repeat the procedure for Lineweaver Burke Plot but this time choose Eadie Hofstee.
a. Note that for this Eadie Hofstee plot the start X value for the linear regression
should be minus 0 and the END X value should be 38000.

COMMON ERRORS - DO NOT TRANSFORM THE Lineweaver Burke data.


You must reselect and transform the Mg-Chelatase data table.
From the linear regression results of these plots calculate the Km and Vmax values (with
errors where possible).
LAYOUT OF THE FOUR GRAPHS
1. CLICK on new layout
2. Choose the layout with 4 boxes as below.

3. Drag the graphs from the left graphs Tab onto the empty boxes on the
layout. Note: You can add annotations to your individual Graphs such
as Km and Vmax values they will automatically appear in the layout.
Make sure your graphs have their axes labeled correctly.
4. Print the layout as a pdf file.
5. Upload this ONE PAGE layout to the Enzyme Kinetics pre-lab
assignment in iLearn prior to the lab class. This ONE PAGE layout is
your pre-lab exercise.

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