Investigating the Effects of Temperature on Enzyme Activity
In this lab exercise, you will investigate enzyme function. Enzymes are large protein molecules (i.e. macromolecules) that act as catalysts in the biochemical reactions that occur in living things. A catalyst is a specific type of protein that increases that rate of a chemical reaction that would otherwise occur to slowly.
How do enzymes increase the rate of a reaction? They lower the amount of activation energy that is needed to get the reaction started.
In an enzyme-catalyzed reaction, the substance that the enzyme acts upon is known as the substrate. Each enzyme is highly specific for a particular substrate because the enzyme has a special area the active site. The active site has a unique shape, and like pieces of a puzzle, the enzyme’s active site can only bind to a substrate with a complimentary shape. When the correct substrate binds, it forms a union known as the enzyme-substrate complex, in which the enzyme begins to change the substrate from its original form. After a short period of time, the substrate is turned into a new product; the enzyme will be recycled to find a new substrate. This process is summarized below:
E + S  E-S  E + P
Thus, it is the highly specific three-dimensional structure of an enzyme that will determine its ability to function properly. However, there are environmental conditions that can change the shape of an enzyme. When this happens, we say that the enzyme has become denatured, and its activity decreases and possibly stops working all together. There are two factors that can alter the shape of (i.e. denature) enzymes: pH and temperature. An enzyme’s optimal pH and temperature is where it works most efficiently. For most enzymes, an optimal pH is 7.4 (i.e. neutral conditions) and an optimal temperature is 37 o C. For these enzymes, they become denatured at extremely low/high pH’s (i.e. acidic conditions, pH 0-3, and basic conditions, pH 12-14).
Temperatures around 40-50 o C will denature some enzymes, but others won’t denature until they reach 70-80 o C; almost all enzyme activity will cease 100 o C (i.e. boiling).
The enzyme you will investigate is this lab is called catalase. Catalase is found in the tissues of many organisms
(both plants and animals) because it plays a very important role in protecting cells from a harmful chemical called hydrogen peroxide (H
2
O
2
). Hydrogen peroxide is a normal by-product of cellular metabolism; however, it quickly becomes a poisonous toxin if it builds up in the cell. The enzyme catalase is responsible for speeding up the breakdown of toxic hydrogen peroxide into two harmless substances, water and oxygen. This chemical reaction is represented by the following chemical equation:
2 H
2
O
2
 2 H
2
O + O
(catalase)
2
Purpose: To investigate the effects of temperature on catalase activity
Objective: To determine the optimal temperature for maximum catalase efficiency.
Pre Lab Questions:
1. Why are doing this lab?
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2. What is an enzyme? ___________________________________________________________________________
3. What are catalysts? How do they work? _______________________________________________________
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4. What is activation energy? ____________________________________________________________________
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5. What is the most important characteristic of an enzyme? ________________________________________
6. What does it mean to be denatured? __________________________________________________________
7. What can affect enzyme function? Why?
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8. What is the enzyme in this lab and why is it important? __________________________________________
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9. What is a substrate? ___________________________________________________________________________
10. What is the substrate in this lab and why is it important in living things? __________________________
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11. How will enzyme activity be measured? Explain! _______________________________________________
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12. Would the chemical reaction: H
2
O
2
 H
2
O + O
2
work if catalase wasn’t present? Explain!
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*** Important Note: These questions are to help you begin thinking about the “background information” that
you will need to include in the INTRODUCTION section of your laboratory report. Remember to ask yourself,
what terms and concepts are needed in your introduction section to allow someone who is reading your lab
report to completely understand what the experiment was about?
12. Identify the following:
Independent Variable
Dependent Variable
Control Variables
What is your Hypothesis: [remember, if…(independent variable)…, then…(dependent variable)…]
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MATERIALS
 4 large test tubes
 Test tube rack
 Hot plate
 ice water bath
 transfer pipette
 10 mL graduated cylinder
PROCEDURE:
Part I: Ice Water Bath
 Two 250 mL beakers

 3.0% H

Catalase
2
O
2
(Hydrogen Peroxide) test tube holders
 glass bends and rubber tubing
1. Place _____ mL of catalase into a clean test tube.
2. Place this test tube into an ice water bath for five minutes. Record the temperature of the bath.
3. While you are waiting, fill a 250 mL beaker with water.
4. Obtain a rubber test tube stopper that has a glass bend and tubing attached.
5. Submerge the tubing in the 250 mL beaker containing the water.
6. At the end of five minutes, remove the test tube (with the catalase) from the ice water bath.
7. Place 2.0 mL of 3% H
2
O
2
into the test tube with the catalase, then immediately seal the test tube with the
stopper that has the glass bend and rubber tubing attached. (See illustration below)
8. Allow the reaction to proceed for three minutes. Count the bubbles that begin to form in the water over this
period of time. At the end of three minutes, record the total number of bubbles you counted in your data
table. The activity of catalase is measured by counting bubbles.
Part II: Room Temperature
1. Place _____ mL of catalase into a clean test tube.
2. Look at your thermometer and read the temperature (this is room temp, record in your data table).
3. Fill a 250 mL beaker with water.
4. Obtain a rubber test tube stopper that has a glass bend and tubing attached.
5. Submerge the tubing in the 250 mL beaker containing the water.
6. Place 2.0 mL of 3% H
2
O
2 into the test tube with the catalase, then immediately seal the test tube with the
stopper that has the glass bend and rubber tubing attached.
7. Allow the reaction to proceed for three minutes. Count the bubbles that begin to form in the water over this
period of time. At the end of three minutes, record the total number of bubbles you counted in your data
table. The activity of catalase is measured by counting bubbles.

Part III: Hot Water Bath #1
1. Fill a 250 mL beaker with water and place the beaker on a hot plate
2. Bring the water to a temperature of 40 o C (NOTE: The hot water bath must be at a constant temperature of
40 o C. If the bath gets too hot, remove the beaker from the hot plate and wait until the water reaches 40 o C).
3. While you are waiting for the water to reach 40 o C, place _____ mL of catalase into a clean test tube.
4. Once your hot water bath is at 40 o C, place the test tube (with the catalase) into the hot water bath for five
minutes. Record the temperature of the bath (it should be roughly 40 o C, 1-2 o C above/below is okay).
5. While you are waiting, fill a 250 mL beaker with water.
6. Obtain a rubber test tube stopper that has a glass bend and tubing attached.
7. Submerge the tubing in the 250 mL beaker containing the water.
8. At the end of five minutes, remove the test tube (with the catalase) from the hot water bath.
9. Place 2.0 mL of 3% H
2
O
2
into the test tube with the catalase, then immediately seal the test tube with the
stopper that has the glass bend and rubber tubing attached.
10. Allow the reaction to proceed for three minutes. Count the bubbles that begin to form in the water over
this period of time. At the end of three minutes, record the total number of bubbles you counted in your
data table. The activity of catalase is measured by counting bubbles.
Part IV: Hot Water Bath #2
1. Bring the hot water bath from part III to a temperature of 60 o C (NOTE: The hot water bath must be at a
constant temperature of 60 o C. If the bath gets too hot, remove the beaker from the hot plate and wait until
the water reaches 60 o C).
2. While you are waiting for the water to reach 60 o C, place _____ mL of catalase into a clean test tube.
3. Once your hot water bath is at 60 o C, place the test tube (with the catalase) into the hot water bath for five
minutes. Record the temperature of the bath (it should be roughly 60 o C, 1-2 o C above/below is okay).
4. While you are waiting, fill a 250 mL beaker with water.
5. Obtain a rubber test tube stopper that has a glass bend and tubing attached.
6. Submerge the tubing in the 250 mL beaker containing the water.
7. At the end of five minutes, remove the test tube (with the catalase) from the hot water bath.
8. Place 2.0 mL of 3% H
2
O
2
into the test tube with the catalase, then immediately seal the test tube with the
stopper that has the glass bend and rubber tubing attached.
9. Allow the reaction to proceed for three minutes. Count the bubbles that begin to form in the water over
this period of time. At the end of three minutes, record the total number of bubbles you counted in your
data table. The activity of catalase is measured by counting bubbles.
RESULTS:
Data Table: Catalase Activity at Various Temperatures
Part I: Ice Water Bath
Part II: Room Temperature
Part III: Hot Water Bath #1
Part IV: Hot Water Bath #2
Temperature ( o C)
Number of Oxygen
Bubbles in 3 Minutes

GRAPHING YOUR RESULTS:
Use Excel to plot oxygen bubbles produced versus temperature on a graph. Follow the steps below to create your graph using excel: a.
Open excel b.
In column A, enter your x-axis data (independent variable)  these are your temperature values
(ice bath, room temp., hot water bath #1, hot water bath #2) c.
In column B, enter your y-axis data (dependent variable)  these are your bubbles counted numbers d.
Click insert tab e.
Click scatter, then click the very first graph option f.
Your graph should now be visible g.
Click on the graph, then click the layout tab at the top of the screen. h.
Click on “chart title”, choose the “above chart” option, then enter the title for your graph i.
Click “axis titles”, then label each axis appropriately (don’t forget to include UNITS used) j.
Choose the “scatter with smooth lines and markers” option (i.e. the top right option) k.
Click “print” (make sure that everything is labeled first). l.
Save this graph in your H: drive in case you need it later.
Discussion/Conclusion Questions: Use these questions (and their answers) to guide you as you write the DISCUSSION section of your lab report.
1. How did you know that the catalase was working?
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2. If catalase is working properly, what is the liquid substance that is left inside the test tube at the end
of the reaction time? Explain!
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3. What effect does increasing the temperature have on catalase activity? Explain (don’t forget to
CITE SPECIFIC DATA to support any statements you make)
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4. Based on your results and your graph, what is the optimal temperature for maximum catalase
activity? Explain (don’t forget to CITE DATA to support your reasoning).
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5. What happens to enzyme activity when the optimum temperature is exceeded? Give SCIENTIFIC
REASONING as to why this happens (hint: structure)
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6. Which temperature acted as the control in this experiment? ____________________________________
7. In CONCLUSION, what is the importance of enzymes in living things and what do you know how
they function?