Name _______________________

Period ____

AP LAB 2: Enzyme-Catalyzed Reactions

Introduction: Enzymes are proteins that function as biological catalysts. Catalysts are substances that speed up
chemical reactions.
Enzymes either:
Catalyze the breakdown of a substrate molecule into products.
Catalyze the assembly of substrate molecules (monomers) into a larger
product (polymer)
In this lab we will use the enzyme catalase and the substrate hydrogen
peroxide (H2O2) at a concentration of 3%. We will determine the effect of
temperature and pH on the action of this enzyme. Catalase was one of the first
enzymes discovered, and was named after its function a catalyst. Catalase is
an
enzyme normally found in many plant and animal tissues. Catalase is found in most cells, even in singlecelled eukaryotes like yeast. Catalase speeds up the breakdown of peroxides which destroy toxic
substances which may form during respiration (metabolic energy production). Also, some cells use
catalase to destroy cellular debris or worn out organelles. This breakdown prevents the peroxide from
causing unwanted oxidation of important biomolecules:
Catalase works by the following mechanism: 2 H2O2 + Catalase 2 H2O + O2 (gas) + Heat
Notice that Catalase is not changed by the reaction, and that the reaction produces heat. Reactions that
produce heat are called exothermic. Hydrogen peroxide will break down all by itself (spontaneously) at a slow
rate. Catalase makes the reaction thousands of times faster and causes bubbling. It is important to note that there
are several ways that this reaction can be measured, such as measuring the level O 2 gas produced by the
reaction, or to measure the pressure of the O2 gas. We have chosen this method because it is simpler and doesnt
require use of dangerous chemicals like potassium permanganate which is normally used as an indicator of the
activity level of Catalase. Potassium Permanganate will remain pink if there is hydrogen peroxide present in
solution, so it can be used to indicate the activity level of Catalase. If the solution remains pink then Catalase is
not breaking down the H202. A brownish-yellow color indicates that there is less H 2O2 in solution. It is important to
be aware of the alternate ways to test for Catalase activity. All enzymes are proteins with active sites that have a
shape that must exactly fit the substrate. Heat and strong acids can destroy the shape of the active site and make
the enzyme nonfunctional. This is called denaturation.
We will measure the reaction rate visually and assign a qualitative value
Table 1: Reaction Rate
Value
0
1
2
3

Rate
no bubbling
small bubble formation
bubbling fast
rapid bubbling/foaming

However reaction rate can also be measured qualitatively so you will need to calculate the reaction rate by
calculating the volume of each reaction using the equation r2h. To do this, Wait one minute and record the height
of the bubbles in each tube in cm you will also need to measure the radius of the test tube. Record in the included
data tables.
SAFETY---SAFETY---SAFETY---SAFETY---SAFETY---SAFETY
1.
2.
3.
4.

Safety Glasses must be worn over eyes at all times during this lab.
Aprons must be worn since there is a danger of hot water spills.
Liver and hamburger can carry salmonella and E. coli!
Hands must be washed AFTER COMPLETING THIS LAB.

Part I: Where is Catalase Found?

Hypothesis:
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Materials:
transfer pipet
cracker
salt
paper

potato
hamburger
sugar
well plate

carrot
liver
onion
forceps

H2O2

Procedure:
1. Carefully place a small quantity of each material listed above in a well.
2. Do not cross contaminate material WIPE OFF FORCEPS
3. Add 5 drops of H2O2 to each well and compare results to the reaction rate table (pg 1)
4. Make sure to use the same amount of H2O2 to each well
Substance

H2O2 (control)

Potato

carrot

Liver

Hamburger

salt

sugar

onion

cracker

paper

Reaction
rate

Conclusion: (accept or reject hypothesis)

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Analysis for Part I: (Results, Evidence, Explanation, Possible Errors)
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Part II: What Effect Does Heat Have on Enzyme Function?

Hypothesis:
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Materials:
liver

3 test tubes

hotplate

3 beakers
transfer pipets (3)
water

test tube holder

H2O2

Procedure:
1. Carefully place a small booger-sized piece of raw liver in each of three test tubes.
2. Using a transfer pipette -add 4 mL of water to each of the separate test tubes.
3. Place one test tube in the test tube rack. This is your control.
4. Place the other test tube in a beaker of simmering water.
5. Place the other test tube on ice.
6. Let the liver cook in the water for 10 minutes.
7. Drain the water out of each test tube, being careful not to lose the liver pieces.
8. Set the temperature of all test tubes by placing them in a beaker of cold water together for 3 minutes.
9. Add 3 mL of H2O2 to each test tube and compare results to the reaction rate table (pg 1)

Data:
Group
Control (raw)
Experimental (cooked)
Experimental (on ice)

Reaction rate

Table 1: Effect of Temperature on Catalase Reaction

Height of Bubbles (cm)
Radius of Test Tube (cm)
Volume of Reaction (cm3)
Hot
Room Temp
Cold
Conclusion: (accept or reject hypothesis)
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Analysis for Part II: (Results, Evidence, Explanation, Possible Errors)
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Part III: Does acid destroy an enzymes ability to speed up a chemical reaction?
Hypothesis:
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Materials:
transfer pipet
liver

3 test tubes
water

cylinder
thermometer

test tube
holder
H2O2

Procedure:
1. Carefully place 5 drops of liver in each of two test tubes.
2. Add 3 mL of lemon juice to one tube (experimental)
3. Add 3 mL of baking soda soln. to one tube (experimental)
4. Add 3 mL of water to the other test tube. (control)
5. Swirl all test tubes for 3 minutes.
6. Pour off the liquid from each tube
7. Add 3 mL of peroxide to each test tube.
8. Record your observations, and compare results to the reaction rate table (pg 1)

Data
Group
Control (water)
Experimental (lemon juice)
Experimental (baking soda)

Reaction rate

Table 2: Effect of pH on Catalase Reactions

Height of Bubbles (cm)
Radius of Test Tube (cm)
Volume of Reaction (cm3)
Acid
Neutral
Base
Conclusion: (accept or reject hypothesis)
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Analysis for Part III: (Results, Evidence, Explanation, Possible Errors)
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Part IV: Questions

1. Draw a diagram that shows how catalase, H2O2, water, and O2 relate to each other in this lab.

2. Where are catalases found naturally?

3. What is the function of catalase in living cells?
4. Why type of biological molecule is an enzyme?
5. What happened effect does lemon juice have on catalase enzyme?
6. What effect did heat have on the activity of the enzyme?
a. What effect did cold have?
b. Room Temperature?
7. What are the substrates catalase bonds with?
8. What is hydrogen peroxide changed into when catalase reacts with it?
9. Is catalase changed during the reaction?

10. What happens to enzymes when they are heated or exposed to acids?

11. Explain why peroxide bubbles when you put it on a cut.

12. Describe how an enzyme works and its importance to all living organisms.

13. What effect did lowering the pH have on the activity of the enzyme?