Name ___________________________ Assignment 1: Getting to Know Enzyme Lab: Setting Up an Experiment The first screen that appears in Enzyme Lab 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 Enzyme Lab. When you set up an experiment in Enzyme Lab, you will add a buffered solution, sucrose as the substrate, invertase (the enzyme), 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 so you can observe the effect of changing these variables on invertase activity. A simulated visible light spectrophotometer will measure product as it is created. Data are recorded and plotted as a function of product concentration [P] in micromoles (m) versus time (minutes). The data you collect can then be analyzed by several different types of plots that are commonly used for analyzing data for enzyme-catalyzed reactions. Assignment #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 Enzyme Lab 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. Just as real experiments are conducted in a biochemistry lab, the enzyme should always be added to the tube last to prevent the reaction from starting before everything has been added to your test tube. Develop a hypothesis to predict the effect of an increase in temperature on invertase activity: I believe that as temperature increases, invertase activity will (choose one: increase, decrease, stay the same) _____________________ because ______________________________________________________
________________________________________________________________________________________. Experiment #1 Set – up:
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 the 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. 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? _________________________________________________________________________________________...
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