Assignments for non-majors

The following assignment is designed to help you become familiar with the operation of EnzymeLab. For your ease in completing each assignment, the background text relevant to the experiment that you will perform is italicized, instructions for each assignment are indicated by plain text, and questions or activities that you will be asked to provide answers for are indicated by bold text.

 

Assignment 1: Getting to Know EnzymeLab: Setting Up an Experiment

The first screen that appears in EnzymeLab shows you a biochemistry lab containing all the reagents and equipment you will need to perform your experiments.

Click on each item in the lab to learn more about its purpose. Once you are familiar with the lab, click on the Experiment button to begin the first assignment. This assignment is designed to help you become familiar with the operation of EnzymeLab.

When you set up an experiment in EnzymeLab, you will add a buffered solution, sucrose as substrate, invertase, and, in some reactions, inhibitors to a test tube to measure the rate of invertase activity. You will have the choice of performing each reaction at different temperatures and under different buffer conditions in which you can examine the effect of these parameters on invertase activity. A visible light spectrophotometer will measure product formation by measuring the absorbance of glucose (released as sucrose is cleaved by invertase) at approximately 450 nm. Data are recorded and plotted as a function of product concentration [P] in micromoles (m m) versus time (minutes). Raw data you collect can then be analyzed by several different types of plots that are commonly used for analyzing kinetic data for enzyme-catalyzed reactions.

1. Effect of Temperature on Invertase Activity

Changes in temperature can dramatically influence the activity of most enzymes by affecting enzyme structure. This exercise is designed to help you learn how to set up an experiment in EnzymeLab and understand the effect of temperature on enzyme activity. You will also analyze data from this experiment to determine the ideal temperature optimum for invertase activity.

To begin any experiment, you first need to set the temperature of your water bath, then add buffer and substrate to the reaction tube. As is the case with real experiments in a biochemistry lab, enzyme should always be added to the tube last to prevent the reaction from starting before all necessary components have been added to your test tube.

Develop a hypothesis to predict the effect of an increase in temperature on invertase activity, then test your hypothesis as follows.

Notice that the default temperature for the water bath is 40° C. To change the temperature, you can either enter a temperature value in the text box or use the arrows. Change temperature to 30° C; this is the lowest temperature at which you can carry out an experiment. Notice that the default buffer pH is 7.0. Do not change the default pH for any measurements in this experiment.

Notice that the default value for substrate concentration [S] is indicated by the slider bar labeled [S] and the value of 25 mM appears in the text box to the far right of the [S] slider. Substrate concentration for sucrose is reported in millimolar (mM) units. You can change [S] by either moving the slider bar or typing a value in the [S] text box. For the first reaction in this experiment we will begin by carrying out a reaction with 90 mM sucrose. Change [S] to 90 mM. Do not select any inhibitors for this experiment.

Note: The [S] value that you use for any experiment will be reported in the table under the Plot Data view. However, two students running an experiment with the same [S] may see slightly different results because EnzymeLab is programmed to simulate differences that represent slight experimental variations in concentration measurements such as you would encounter if setting up this experiment in a wet lab.

To add enzyme to your reaction tube, click the Add Enzyme & Go! button. This will also activate the spectrophotometer to measure product concentration.

a. Determining Starting Velocity (VO)

After each time you add enzyme, a plot of product concentration versus time will appear with enzyme kinetic data plotted as data points in solid black circles.

What did you observe for the plot of product concentration versus time? Is this what you expected? Explain your answer.

To begin your analysis of this experiment, you first need to determine the starting velocity of the reaction (VO). This is easily accomplished because VO (the initial rate of the reaction showing first-order kinetics where the rate of the reaction depends on [S]) is represented by the slope of the linear portion of the curve in this type of plot. The plateau (asymptote) of the plot represents zero-order kinetics where the rate of the reaction does not depend on [S]. Note: For many of the reactions you will run, the reaction will not reach zero-order kinetics.

To determine the slope of the line, a red line will appear on the plot. You can then click on this red line and move it to find the best fit for the slope of the plotted points for invertase activity. Try to align the red line so that you have an equal number of data points bisected by the line and an equal number of points above and below the line. This is generally a good approach for finding the best-fit slope. The program will not tell you when you have found the best fit, so use your best judgment! Follow the directions below to determine the slope of the line for this first measurement.

Click within the plot and move the red line until you have found the slope of the plotted data points. Before we can analyze this information further, you must record your data by clicking the Record Data button. Note: The Record Data feature is not active until you have properly determined the slope of the plotted line.

After you have recorded data for a measurement, you must click the Clear Experiment button before you can take another measurement. Note: If you forget to record data and attempt to clear the experiment, a warning box will appear asking if you want to clear data without recording it.

Keeping buffer pH constant and [S] at 90 mM, create another experiment but this time increase temperature to 35° C. Run the experiment, determine the slope of the line, then record this measurement. Repeat this process to set up experiments increasing temperature in 5-degree increments (40° C, 45° C, 50° etc.) until you reach the maximum temperature of 85° C. Find the slope of the line for each experiment and record these data.

Click on the Plot Data button to prepare a plot of your data for this experiment.

b. Plotting Invertase Kinetic Data

In the first window that appears in the Plot Data view you can title the plots for the experiment you are working on, select a plot to create, change the symbols for this plot, and view raw data in tabular form showing each measurement for a particular experiment–[S], the presence or absence of an inhibitor, inhibitor concentration [I], temperature, pH, and VO.

From the Plot Data view we can now use EnzymeLab to carry out a number of important calculations and present these data as plots that are traditionally used for studying enzyme kinetics. Each of the plots available in EnzymeLab is briefly described below. It is very important that you are comfortable with the abbreviations referred to below and the purpose of plotting data with these different types of plots. These principles form the basis for the majority of experiments that you will be carrying out with EnzymeLab.

VO vs. [S]: convenient way to express the relationship between reaction velocity (Vo) and substrate concentration [S]. Used to determine Vmax, the maximum velocity of an enzyme, represented by the asymptote (plateau) of the line. We can also use this plot to measure the Michaelis constant (KM). To find the Michaelis constant, you need to locate -Vmax then find where this value would intersect the x-axis. The [S] represented by this intersect point is the KM. This type of plot is a simple way to represent and determine Vmax and KM; however, it is not the most accurate way to determine these values because we are extrapolating from the plot.

VO vs. Temperature: analyzes the effect of temperature on reaction velocity.

VO vs. pH: analyzes the effect of pH on reaction velocity.

Lineweaver—Burk: also called a double-reciprocal plot. Produces a linear plot for the inverse of velocity (1/V) versus the inverse of substrate concentration [1/S]. Used to determine two important characteristics of enzymes that follow Michaelis—Menton kinetics: KM (substrate concentration at which a reaction has reached half of its maximum velocity), and kcat (turnover number = number of substrate molecules undergoing a reaction per enzyme molecule per second). The Lineweaver—Burk plot is a more accurate plot for determining Vmax and KM than a plot of VO versus [S] because Lineweaver—Burk plots are based on algebraically arranged equations of the Michaelis—Menten equation.

Eadie—Hofstee: a plot of V versus V/[S]. Another way to determine parameters of Michaelis—Menton kinetics. The y-intercept indicates Vmax, the x-intercept determines Vmax/KM, while the slope of the line determines -KM.

Note: Any of the plots that you generate can be saved to disk or printed by clicking on the Export Graph button, which appears to the left of each plot. Clicking on this button will open a separate window with your plot. From this window you can then save your plot to your hard drive or a disk, and you can print your plot by using the print feature of your browser software.

Plotting VO vs. Temperature: Click in the Title box and type in the title "Experiment 1 — Temperature Optimum." VO vs. [S] should appear as the default plot in the Plot Type box. Click on the popup menu and select VO vs. Temperature. For any plot, you must first select the data that you want to plot. You can click on an individual row to select it and the row will be highlighted, or you can select several rows by holding down the Shift key and clicking on each row. Shift-click on each row of measurements that you recorded for this temperature experiment, then click the Plot Selected Data button to produce a plot of VO versus temperature.

Click anywhere on the VO vs. temperature plot and drag the vertical gray dashed line to locate the highest value for enzyme activity (Vmax). When you have correctly located this value, the gray line will become black and it will freeze in place. This value, indicated in the best-temperature text box, represents the optimal temperature for invertase activity under these conditions of pH and [S].

What is the optimal temperature for invertase activity? Is this what you expected? Would invertase isolated from any two organisms (for example, yeast invertase vs. invertase from the small intestine of humans) show the same temperature optimum? Why or why not? Explain your answers.

Explain why temperatures lower or higher than the optimum cause decreases in invertase activity. What is happening to the enzyme to produce these decreases in activity?

Carefully examine the curve for VO vs. temperature. Is the slope of the line on both sides of the curve the same or different? If the slope of the line to the left of maximum velocity is different from the slope of the line to the right of maximum velocity, explain why this is. What is responsible for these differences in enzyme kinetics?

If you were to carry out these temperature experiments at a higher or lower [S], what effect would [S] have on the temperature optimum for invertase? Formulate a hypothesis and then test your hypothesis. What did you discover? Explain your results.

If you were to carry out these temperature experiments at a higher or lower pH value, what effect would this have on the temperature optimum for invertase? Formulate a hypothesis and then test your hypothesis. What did you discover? Explain your results.