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Name:_______________________________
Biology
Date:__________________
LAB: Investigating Enzyme Function (Part 1)
Objectives:
- What is the role of enzymes in chemical reactions?
- Can enzymes be reused?
- How can you demonstrate that enzymes can/cannot be reused?
Materials:
test tube rack
5 test tubes
liver 3% hydrogen peroxide
goggles
Safety: Goggles must be worn. Wash hands after handling liver. Do not ingest solutions or eat liver.
Background Information
At any given moment, all of the work being done inside any cell is being done by enzymes. The purpose of an
enzyme in a cell is to allow the cell to carry out chemical reactions very quickly. Enzymes are proteins that speed
up the rate of reactions that would otherwise happen more slowly. These reactions allow the cell to build things or
take things apart as needed.
Enzymes are made from amino acids. When an enzyme is formed, it is made by stringing together between 100
and 1,000 amino acids in a very specific and unique order. The chain of amino acids then folds into a unique shape.
That shape allows the enzyme to carry out specific chemical reactions -- an enzyme acts as a very efficient catalyst
for a specific chemical reaction.
For example, the sugar maltose is made from two glucose molecules bonded together. The enzyme maltase is
shaped in such a way that it can break the bond and free the two glucose pieces. The only thing maltase can do is
break maltose molecules, but it can do that very rapidly and efficiently.
substrate
In this lab, you will study an enzyme that is found in the cells of many living tissues. The name of the enzyme is catalase
(KAT-uh-LAYSS); it speeds up a reaction which breaks down hydrogen peroxide, a toxic chemical, into 2 harmless
substances--water and oxygen. Hydrogen peroxide is a product of cellular activities but needs to be broken down because
it is poisonous to cells. The reaction is as follows:
CATALASE
2H2O2 -----------------------> 2H2O + O2
Reactant
Hydrogen peroxide
Products
Water + Oxygen
Pre-Lab Questions
1. The reaction is catalase facilitating is : _______________________________________
2. Why is this reaction necessary in the body?
3. The enzyme in this reaction is ________________ & the substrate is _______________
4. The substrate fits into a location of the enzyme called the _______________________.
5. The reactants are _______________________ & the products are ________________.
Pre-Lab Procedure and Discussion Points:
1. Place 2 pipettes of hydrogen peroxide (1 mL) in a small clean test tube and put the test tube in the rack.
2. Add a small piece of enzyme (liver) to the peroxide.
3. Record observations in Table 1 (New H2O2 + New Catalase)
4. Feel the bottom of the test tube and note any temperature change. What might be the source of the
change? What are some possible explanations for the bubbles.
Objectives: How do enzymes work and can enzymes be reused?
Hypothesis:
To explain this reaction, three hypotheses are possible. Choose ONE:
a. Catalase and hydrogen peroxide together form one or more new products.
b. The hydrogen peroxide is changed in the presence of the enzyme, but the enzyme does not change.
c. The enzyme is changed in the presence of hydrogen peroxide, which does not change.
Table 1. Predictions & Results:
Reaction
Prediction
(bubbles or no
bubbles?)
New H2O2
+
New Catalase
Used H2O2
+
New Catalase
New H2O2
+
Used Catalase
New H2O2
+
New, Cooked Catalase
Observations
Explanation
Analysis:
1.
What role does catalase play in a cell? Is this enzyme found only in the liver? (HINT: Hydrogen peroxide will bubble
if placed on a cut finger. What does this tell you?)
2.
Carbon dioxide is also produced by reactions in living cells. Does catalase cause the same reaction with carbon
dioxide or carbonic acid as it does with hydrogen peroxide? (Carbonic Acid is formed when carbon dioxide dissolves
in water.) Why or why not? (Consider how enzymes work).
3.
If left open in the sunlight, a bottle of hydrogen peroxide will slowly change into water and oxygen. Under these
conditions it takes a very long time for the reaction to happen. On the other hand, when catalase is placed in the
hydrogen peroxide, the reaction takes place almost immediately. What does this tell you about the function of the
enzyme regarding the breakdown of hydrogen peroxide in living systems?
Conclusion: * Formal scientific writing has no personal pronouns and is written in the past tense.
A Conclusion Paragraph usually contains a description of the purpose of the experiment, a
discussion of your major findings, an explanation of your findings, and recommendations for further
study. Usually the following questions are presented in paragraph form:
1. What was the purpose of the experiment?
Format: The purpose of the experiment was to investigate (Insert Title.)
Example: The purpose of the experiment was to investigate the effect of stress on the growth of bean plants by
comparing the growth of bean plants subjected to stress for 15 days with a control (non-stressed plants.)
2. What were the major findings?
Format: The major findings were (Insert Results Sentence.)
Example: The major findings were that there was no significant difference existed between the mean height of
stressed plants and non-stressed plants 30 days after transplanting.
3. Was the hypothesis supported by the data?
Format: The hypothesis that (Insert Hypothesis) was (supported, partially supported, or not supported.)
Example: The hypothesis that stressed plants would have a lower mean height was not supported.
4. How did your findings compare with those of researchers/lab groups?
Example: In contrast, Japanese farmers found that hitting and pulling rice plants were beneficial to plant
height.
5. What happened that you did not expect? How can you explain this?
Format: This was unexpected. (Insert anomaly if there was one.) This can be explained by (insert explanation.)
Example: The stressed bean plants were expected to have a lower height. The fact that they didn't and that
Japanese rice farmers stress their points on purpose to achieve better growth means that something about
stressing out plants makes them growth better. Perhaps some plants that are stressed release a chemical in
response to the stress that promotes better growth and others don't release that chemical, such as rice vs. beans.
Or perhaps there is a difference in reaction to stress between monocots and dicots.
6. What recommendations do you have for improving this experiment?
Example Improved experimental design techniques including a larger sample and a longer growing period would
benefit a similar study.
7. What recommendations do you have for further study? (This is above and beyond this experiment.)
Example: Additional investigations using various sources of stress at more frequent intervals would be a good
additional experiment. another idea would be to use different types, such as a monocot and a dicot. If further
research were done, perhaps scientists have isolated a chemical released by plants during stress. It would be
interesting to investigate the amounts of this chemical released during stress.
Name:_______________________________
Biology
Date:__________________
LAB: Determining the Optimal Conditions for the Enzyme Catalase (Part 2)
Objectives:
 What are the optimal temperature and pH conditions for catalase function?
 What conditions can cause a decrease in catalase function and why?
Hypothesis: Write a hypothesis for each experimental (independent) variable in “If/then/prediction of observed
result” format.
 Temperature Hypothesis:

pH Hypothesis
Materials
Liver
HCl
solution
3%
hydrogen
peroxide
NaOH
solution
6 test tubes
& rack
Goggles
3 beakers
Indicator
(pH paper)
ice
Hot hands/
clamps
Thermo-meter
Safety: Goggles must be worn with glassware; Wash hands after handling liver. Gloves must be worn to handle acidic
and basic solutions.
Procedure
Determining Optimal Temperature Conditions for Catalase
Room Temperature: Make a “Baseline Observation” for rate of reaction at room temperature.
1. Place 2 ml of the 3% hydrogen peroxide solution into a clean test tube.
2. Add a small piece of liver to one test tube. Observe the bubbles.
What gas is being released? _______________________________________________
3. Throughout this investigation you will estimate the rate of the reaction (how rapidly the solution bubbles)
on a scale of 0-5 (0=no reaction, 1=slow,...., 5= very fast). Assume that the reaction in step 2 proceeded at a
rate of "4" and record the speed in the DATA TABLE as the rate at room temperature.
Boiling (100 C)
4. Put a piece of liver into the bottom of a clean test tube and cover it with a small amount of distilled water. Place
this test tube in a boiling water bath for 5 minutes. What effect do you think boiling will have on the enzyme?
Provide a reason for your answer.
___________________________________________________________________________________________
_____________________________________________________________________________
5. Remove the test tube from the hot water bath, allow it to air cool, then pour out the water. Add 2 ml of hydrogen
peroxide. CAUTION: Use a test-tube holder when handling the hot test tubes.
What is happening in the test tube? ___________________________________________
Record the reaction rate (0-5) the DATA TABLE.
Ice Bath (0 C) and Warm water bath (37 C)
6. Put equal quantities of liver into 2 clean test tubes and 1 ml H2O2 into 2 other test tubes. Put one test tube of liver
and one of H2O2 into each of the following water baths: Ice bath (0 deg.C) and Warm water bath (37 deg.C)
7. After 3 minutes, pour each tube of H2O2 into the corresponding tube of liver and observe the reaction. Record the
reaction rates (0-5) in the DATA TABLE.
o
What is the "optimum" temperature for catalase? (This is the temperature at which the reaction proceeds
fastest.) ____________________________________________________________________________
o
How was the rate of reaction affected by cooling to 0 degrees C? Provide an explanation for your
observation.
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
How was the rate of reaction affected by heating to 100 degrees C? Provide an explanation for your
observation.
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
o
Determining the optimal pH for catalase activity: Teacher Station
1. Add 2 ml hydrogen peroxide to each of 3 clean test tubes. Treat each tube as follows:
Tube 1--add a drop of 1molar HCl (acid) at a time until pH 3.
Tube 2--add a drop of 1molar NaOH (base) at a time until pH 10.
Tube 3--adjust the pH to 7 by adding single drops of either 1molar HCl or 1molar NaOH as needed.
CAUTION: Do not let acids or bases contact your skin or clothing. Swirl each test tube after adding each
drop and measure the pH of each solution with pH paper. To do this, remove a drop or two of solution
from a test tube using a clean glass stirring rod. Rinse your stirring rod and wipe dry before you dip it into
each test tube. Place the drop on pH paper. Record the pH of each solution in DATA TABLE 1.
2. Next, add a small piece of liver to each test tube. Estimate the reaction rates (0-5) and record in DATA TABLE 1.

Does there appear to be a pH "optimum"? At what pH? Provide evidence to support your answer.
_______________________________________________________________________________
_______________________________________________________________________________
Data Table: Catalase Activity at different Temperatures and pH
Temperature
Ice bath (0 C)
Room Temp. (22 C)
Warm water (37 C)
Boiling Water (100 C)
Enzyme Activity
(0 none 5 very fast)
0 1 2 3 4
5
0 1 2 3 4 5
0 1 2 3 4 5
0 1 2 3 4 5
0 1 2 3 4 5
pH
3 (acidic)
7 (neutral)
10 (basic)
Enzyme Activity
(0 none 5 very fast)
0 1 2 3 4 5
0 1 2 3 4 5
0 1 2 3 4 5
Lab Write Up
Your lab write up should have the following sections (in this order). Please use your Experimental
Design Reference packet (and its checklists) as you prepare your lab report.








Title
Hypothesis (Make sure you address temperature and pH conditions.)
Procedure
Data Table
Graph
Results sentence
Summary
Conclusion