One of the most fundamental kinds of experiments utilized when investigating the properties of enzymes is an **Enzyme Assay**, used to measure **Enzyme Activity **(how well the enzyme is able to catalyze a reaction) under different conditions. Often, a **Time Course **is used, in which the investigator measures how fast the enzyme catalyzes the reactions over a set amount of time.

For this exercise, we will be using data that have been collected by actual students working in the lab with the enzyme **Lactase, **looking at how 5 different factors affect the rate at which the product of the reaction is formed. Please see the “**Enzyme Lab Procedure and Worksheet**” handout for the background information and details of how this experiment is done in the laboratory. It is an exciting, hands-on lab, usually done over the course of 2 weeks, which requires the student to learn the use of **micropipettes **and the **spectrophotometer **to measure enzyme activity. Whereas this exercise cannot replace the experience of working in the lab, it does provide an opportunity to become familiar with the techniques employed, and to work with real data to visually display and analyze how an enzyme responds to changes in conditions such as pH, temperature, substrate concentration and inhibitors.

After reading through the Procedure to help you understand the type of data collected, use the data tables supplied in this handout to create 4 or 5 (5^{th} graph is extra credit!) graphs showing the time courses used to measure enzyme activity (part “b” of each activity). As described in the Procedure, once you graph these time courses in Excel, or other spreadsheet program, you will need to do **linear regressions **(also called “**Trend Lines**” in Excel) to obtain the **Rate of Product Formation **for each treatment. The rate of the reaction is simply the slope of the linear regression line. You will then use these rates to generate a second set of graphs (part “d” of each activity) which more clearly show the effects of pH, temperature, etc. on enzyme activity.

Please submit your graphs as a single file uploaded in Canvas, along with the tables (part “c” of each activity) that you have filled in with the values obtained from your Trend Line graphs.

The Worksheet that can be found at the end of the Procedure is intended as an aid in learning the concepts associated with how enzymes work and are affected by the factors studied in this experiment. Please go through it before doing the graphs for Part 1 and answering the questions for Part 2 of the lab.

For Part 2, you will answer some fundamental questions about measuring enzyme activity, in this case lactase (known technically as B-galactosidase), the use of a micropipette, and the units commonly used to measure volumes and concentrations of solutions in enzyme assays.

# ENZYME LAB PART 1: Graphing and Analysis

**NAME: _Instructor: **

** **

**STATION #_ _ Your LAB PARTNER: _ _ Activity #1B. Standardization of Enzyme Solution**

1a. Record your experimental data in the table below.

1b. Plot the data as “Time (min)” on the X axis vs. “Enzyme Activity (Absorbance @450nm)” on the Y axis, using Excel. Use an X-Y Scatter Plot with straight lines connecting the points. **(EXTRA CREDIT)**

1c. Using the “**Trend Line**” function, do a linear regression on each set of points. Check the “**Display equation on chart” **box. Write the slopes of each linear regression equation for each volume of enzyme in the chart below. In each case, the slope is equal to the __rate__ of enzyme activity measured as the change in absorbance per minute (DA_{450}/min).

1d.Now, use the data from the above table to Plot the data as “Enzyme Volume (mL)” on the X axis vs. “Rate of Product Formation (DA450/min)” on the Y axis, using Excel. Use an X-Y Scatter Plot with straight lines connecting the points.

__Activity #2. The effect of pH on Enzyme Activity__

2a. Record your experimental data in the table below.

2b. Plot the data as “Time (min)” on the X axis vs. “Enzyme Activity (Absorbance @450nm)” on the Y axis, using Excel. Use an X-Y Scatter Plot with straight lines connecting the points.

2c. Using the “**Trend Line**” function, do a linear regression on each set of points. Check the “**Display equation on chart” **box. Write the slopes of each linear regression equation for each pH in the chart below. In each case, the slope is equal to the __rate__ of enzyme activity measured as the change in absorbance per minute (DA_{450}/min).

2d. Now, use the data from the above table to Plot the data as “pH” on the X axis vs. “Rate of Product Formation (DA450/min)” on the Y axis, using Excel. Use an X-Y Scatter Plot with straight lines connecting the points.

__Activity 3: Effect of Temperature on Enzyme Activity__

3a. Record your experimental data in the table on the next page.

3b. Plot the data as “Time (min)” on the X axis vs. “Enzyme Activity (Absorbance @450nm)” on the Y axis, using Excel. Use an X-Y Scatter Plot with straight lines connecting the points.

3c. Using the “**Trend Line**” function, do a linear regression on each set of points. Check the “**Display equation on chart” box**. Write the slopes of each linear regression equation for each temperature in the chart below. In each case, the slope is equal to the __rate__ of enzyme activity measured as the change in absorbance per minute (DA_{450}/min).

3d. Now, use the data from the above table to Plot the data as “Temperature (^{o}C)” on the X axis vs. “Rate of Product Formation (DA450/min)” on the Y axis, using Excel. Use an X- Y Scatter Plot with straight lines connecting the points.

__Activity #4: Effect of Substrate Concentration on Enzyme Activity__

4a. Record your experimental data in the table below.

4b. Plot the data as “Time (min)” (X) vs. “Enzyme Activity (Absorbance @450nm)” (Y), using Excel. Use an X-Y Scatter Plot with straight lines connecting the points.

4c. Using the “**Trend Line**” function, do a linear regression on each set of points. Check the “**Display equation on chart” box**. Write the slopes of each linear regression equation for each treatment in the chart below. In each case, the slope is equal to the __rate__ of enzyme activity measured as the change in absorbance per minute (DA_{450}/min).

4d. Now, use the data from the above table to Plot the data as “[ONPG] (mM)” on the X axis vs. “Rate of Product Formation (DA450/min)” on the Y axis, using Excel. Use an X-Y Scatter Plot with straight lines connecting the points.

__Activity 5: Effect of Inhibitors on Enzyme Activity__

5a. Record your experimental data in the table below.

5b. Plot the data as “Time (min)” (X) vs. “Enzyme Activity (Absorbance @450nm)” (Y), using Excel.

5c. Using the “**Trend Line**” function, do a linear regression on each set of points. Check the “**Display equation on chart” box**. Write the slopes of each linear regression equation for each treatment in the chart below. In each case, the slope is equal to the __rate__ of enzyme activity measured as the change in absorbance per minute (DA_{450}/min).

5d. Now, use the data from the above table to plot a **column **graph showing reaction rate at each of the different treatments. You may need to consult with your instructor on how to do this. The X axis should be labeled “Treatment” with a column for each treatment.

The Y axis should be labeled the same as the other trend line graphs: “Rate of Product Formation (DA450/min)”