Lab 6 Information

I expect that you will find that this lab has the most complicated data analysis of all the labs that you have done. The procedures used in the lab are fairly straightforward – as are the experimental goals. You will not be doing section V.

Educational Goals:

The goals of this lab include understanding the factors that affect the ability of enzymes to catalyze a reaction. Since enzymes are proteins, it should not surprise you that the activity of enzymes is altered by factors that may lead proteins to denature. These include pH and temperature. In addition, as described in your lab manual, the rate at which an enzyme catalyzes a reaction is also affected by the amount of substrate. These are the factors that you will alter in this lab to measure their effect on the rate of the reaction by the enzyme.

In addition, this lab requires complicated data analysis in order to demonstrate the affect of these various factors on the rate of the reaction.

Experimental Goals:

The goal of this experiment is to determine the effect of substrate concentration, pH and temperature on the rate of reaction of the enzyme glucose oxidase.

Before Lab:

It is expected that you complete the pre lab prior to coming to lab. This completing the calculations needed to set up the serial dilution that you will use to prepare the various glucose concentrations.

You should come into the lab prepared to begin making the serial dilution of glucose. When setting up for the effect of temperature, you should prepare a cuvette (containing the substrate) and a test tube containing the enzyme and let both incubate (do not mix them) in each of the appropriate temperatures. Thus you should have a cuvette and a test tube equilibrating in the 65ºC water bath; another cuvette and test tube in the 37 ºC water bath; another set in the refrigerator (instead of ice); an finally a fourth set should be set out at room temperature.

Make sure that you DO NOT mix the glucose solutions with the enzyme until you are actually ready to put the combined mixture into the spec 20 and measure the absorbance.  NEVER mix more than one tube at a time and do not mix the next one until you have recorded all absorbances from the preceding tube.  If you make this error, your instructor will have no choice but to scream in agony and jump out the window.

After Lab:

1. For every experiment, you must determine the slope of plot of time (x-axis) vs. absorbance (y-axis) as described in you lab book.  All slopes will be determined in this manner.  Of course, you are to use Excel to determine the slopes and r2 (for each set of data).   Do a trendline on the graph for each set of data - you will then determine the slope, the slope is the rate of reaction.  This must be done separately for each set of data and for each different experiment!

2. Prepare one graph for each condition plotting time vs. absorbance  (concentration, pH, temperature and substrate, if done) with each graph containing a plot for each different value.  Thus, there should be one plot for each concentration (there are 6 different concentration) on a single graph dealing with the effect of concentration on the reaction rate.  There should be additional graphs dealing with pH, temperature and substrate (if done).

3. Prepare a table for each different variable (substrate concentration, pH, etc.) showing the value of the variable (each concentration, for example) and the respective the rate of reaction.

4. You then need to prepare a secondary plot graphing the concentration (and the pH and the temperature - three different graphs) vs the rate of the reaction.

Sample Data Analysis:

Figure 1 shows the effect of varying the substrate concentration on the rate of formation of the product (remember, the absorbance is changing due to the formation of hydrogen peroxide, which is a product).

It should be observed that the slope of these lines increases as the substrate concentration increases. This is easily observed when one looks at the slope term in the equations. Note that the r2 indicates that each trendline is linear as it is above 0.90. If your data does not fill this requirement, you may remove the longer time points until the remaining data is linear. The slopes, as a function of substrate concentration is shown in Table 1.

Table 1: Effect of Substrate Concentration on Rate of Reaction

Substrate Concentration (mM)

Rate of Reaction (absorbance/s)

0.10

0.002

0.50

0.003

1.00

0.005

1.50

0.007

This is then plotted to give Figure 2, a graphical representation of the data in Table 1..

 

Real Data:

The following data was from a paper published in Applied and Environmental Microbiology in 2006.  In this paper, they were investigating the enzyme lysine 2,3-aminomutase (KAM).  The figure below shows the effect of enzyme concentration on the activity of the enzyme.  This is a similar to the experiment shown in Figure 1 above.

The activity of KAMPg is dependent upon the concentration of each electron transfer protein. Various concentrations of FPR (A), FD (B), and FLD (C) were used. The concentration of KAMPg was 5 μM. All assays were performed as described in the text and were repeated at least three times. The lines through the data points do not represent the fit of a kinetic model; they are included only for clarity.

 

The following graph was taken from a paper published in Journal of Biological Chemistry in 2006.  This figure shows the effect of pH on the reaction rate of two different forms of Fructose-1,6-bisphsosphate aldolase.  This figure is similar to Figure 2 above.  You should be able to identify at which pH is best for each form.  Which form is more sensitive to changes in pH?