Lab #4: Enzymes & Experimental Design
OBJECTIVES:
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To continue to practice and apply hypothesis testing.
To continue to practice experimental design.
To gain a better understanding of enzymes and some conditions (temperature, pH, and enzyme
and substrate concentration) that affect enzyme activity and the rate of an enzyme-catalyzed reaction.
To learn to use the spectrophotometer
To learn the concepts behind the workings of the spectrophotometer.
General Procedures
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You will work in groups of 4 but all students will complete their own lab questions/data sheet &
turn it in individually.
INTRODUCTION
Now that we have spent some time considering the steps scientists go through in “doing science”,
we’ll try our hands at formulating our own hypotheses, and designing experiments to test them.
Unlike other labs you have done, this will not be a “cookbook” exercise. You will be writing your
own protocol. To do this effectively, you must read this lab thoroughly, and complete the prelab
assignments, before coming to class!
The subject of our inquiry will be enzymes and the various factors that influence their activity.
Without enzymes, most biochemical reactions would take place at a rate far too slow to keep pace
with the metabolic needs and other life functions of organisms. Enzymes are catalysts that speed up
chemical reactions but are not themselves consumed or changed by the reactions.
The cell's biological catalysts are proteins. These enzymes have a very complex three-dimensional
structure consisting of one or more polypeptide chains folded to form an active site-a special area
into which the substrate (material to be acted on by the enzyme) will fit.
Changes in temperature, alterations in pH, the addition of certain ions or molecules, and the presence
of inhibitors all may affect the structure of an enzyme's active site and thus the ability of the enzyme
to catalyze the reaction (“enzyme activity”), and hence the rate of the reaction in which it
participates. The rate of an enzymatic reaction can also be affected by the relative concentrations of
enzyme and substrate in the reaction mixture.
During this exercise you will study the activity of the enzyme catecholase contained in some fruits
and vegetables. Peeled potatoes and bruised fruits turn brown when exposed to air because
catecholase facilitates a reaction between catechol and oxygen. In the presence of oxygen, the
compound catechol is oxidized by the removal of two hydrogen atoms. Catechol is thus converted
to benzoquinone, and oxygen is reduced by the addition of two hydrogen atoms to form water.
Benzoquinone molecules then link together to form long branched chains. These chains are the
structural backbones of the red and brown melanoid pigments that cause darkening. Keep in mind
that whenever you use potato juice in the following experiments, you are using an enzyme
preparation (in which the enzyme is catecholase).
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PREPARATION
Before lab, read over this lab and material in the book on enzymes. Generate a list of variables, or
factors that might affect the rate at which an enzyme can catalyze the conversion of substrate to
product. Think about how you might test the effects of some of these variables. In the space
provided on the sheet at the end of the lab, select one of the variables you identified, and outline the
protocol you would use to test its effects. (You may want to coordinate with your lab group in
selecting a variable, but each student needs to write out their own protocol before coming to
lab.) Think carefully about how many conditions you might want to test in your experiment, and
what type of controls you might need to use to validate your results. Be sure to consult the sample
protocol below as you plan your experiment.
PROCEDURES
Work in groups to decide on one variable for your group to test. Use the sample protocol outlined
below to help you design your group’s experiment. Fill in Table 2 with the hypothesis, prediction
and protocol for your group. Your instructor will consult with each group to approve their protocol
before you begin your experiment!
Note: Be sure to turn on your Spec 20 before your experiment as it will need 10-15 minutes to
warm up!
MATERIALS (PER GROUP)
6 test tubes
1 test tube rack
MATERIALS (TO SHARE)
pH 7 phosphate buffer
potato extract, with pipets
catechol, with pipets
distilled water
6 pieces of Parafilm
3 pipets
50 °C water bath
ice
HCl and NaOH solutions
pH Indicator Paper
spectrophotometer
extra pipets
wax pencils
Kimwipes ®
thermometers
SAMPLE PROTOCOL (Use this protocol as a guide in writing your own!)
1. Identify the variable you wish to explore, and establish the conditions under which you will test
the activity level of our enzyme, catecholase. For instance, if you choose to explore the effects of
temperature, you’ll need to decide on the exact temperatures you wish to test, and set-up the
appropriate ice baths or warm water baths. You should test your enzyme under three conditions.
For example, your group may choose to test your enzyme at 4 °C, 24 °C, and 50 °C. Be aware that
you may need to adapt your experiment to fit the time and supplies available in the lab.
2. Label three tubes with a wax pencil so that you can distinguish one treatment from the next.
Prepare the tubes, each containing 3 ml of pH 7 phosphate buffer, and 0.5 ml of potato juice (our
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enzyme!), and 0.5 ml of water (use a different pipette for the buffer, water and the potato juice).
These will be the blanks for the three conditions you decide to test.
3. Fill three additional test tubes with 3 ml of pH 7 phosphate buffer, and 0.5 ml of potato juice.
Label these tubes such that you can distinguish them from your blanks. These are your
“experimental” tubes.
4. You will have a bottle of catechol, the substrate. When you are ready to begin your experiment,
you can add catechol directly from the bottle using a new pipette for measuring.
5. With your experimental conditions established, add 0.5 ml of catechol to each of your
experimental tubes. (Water has been used to replace this in your blanks.) Cover each tube with
Parafilm and invert it several times to mix the contents. Place the test tubes in their experimental
conditions.
6. Allow the reaction to proceed for 6 minutes. In the meantime, prepare to take a reading on the
Spectronic 20 (“Spec 20” for short). You should be sure to turn on your Spec 20 before your
experiment as it will need 10-15 minutes to warm up! Be sure the wavelength is set to 420.
Adjust the Spectronic 20 to total absorbance, or zero transmission using the zeroing knob (on/off) on
the left. Turn it until the needle aligns on top of the  line, just to the left of “2” on the absorbance
scale. This is called “zeroing” the Spec. (See Figure 1.)
Figure 1: Baush & Lomb, Spectronic 20
7. After 6 minutes, use the blank for your first
treatment to adjust the machine to 0%
absorbance using the blanking knob. This is
called “blanking” the Spec 20. Wipe
condensation off the tube before reading. Then
read the absorbance of your experimental tube
and record your reading in Table 1.
8. Repeat for the other 2 experimental tubes.
Remember to use appropriate blanks at each
condition you test.
CLEAN UP
When done, please empty all tubes in the sink, rinse them out and place them in your test tube rack.
Return all supplies to the front of the room. Throw away your pipets, pieces of Parafilm, and any
papers. Wipe off your desk
DATA ANALYSIS
1. Graph the data from Table 1 (use graph paper). Be sure to label the x- and y-axes appropriately.
Think carefully about what type of graph might be most appropriate for the data you have generated.
2. Fill in the rest of Table 2.
 Based on the data, did you support or reject your hypothesis?
 Explain your reasoning for your conclusion: why did you choose support or reject?.
3. Identify the optimal condition for the enzyme activity.
4. Anser the rest of the questions on the Data Sheet.
5. Be prepared to present your results to the class.
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