SBI4U Factors Influencing Enzyme Activity

Introduction:
Enzymes act as specific catalysts for chemical reactions in living cells, by lowering the activation energy
required for a reaction to occur. The enzyme to be used in this lab is called catalase. This is an enzyme
used to decompose hydrogen peroxide (H2O2), a toxic waste product of metabolism.
catalase
2H2O2 (aq) 2H2O (l) + O2 (g)

In this experiment, the effect of various factors upon the rate of catalase activity will be studied.

Question: How do changes in temperature, pH and enzyme concentration affect the rate of enzyme
activity?

Hypotheses:

Materials:
- 100 mL graduated cylinder - 1.0 M HCl and 1.0 M NaOH - hot plate
- 2 x 10 mL graduated cylinder - 500 mL beaker (for water bath) - crushed ice
- 5 test tubes - test tube tongs or hot gloves - test tube rack
- 100 mL beaker - 3% H2O2 solution - distilled water

- thermometer - prepared diluted liver puree

Please label (using tape) the base of one 10 mL graduated cylinder H2O2 and the base of the
other 10 mL graduated cylinder Liver. This will prevent you from mixing up the solutions!

Safety: Hydrogen peroxide is corrosive and may cause burns to the respiratory tract, skin and
eyes. Do not inhale vapors. Always wear safety goggles. NaOH and HCl are corrosive so
avoid direct contact with these chemicals.

Procedure:

Teacher Preparation:
Your teacher will prepare a stock solution for the class to share as follows: 150 g of beef liver pureed
with 1 L of distilled water. Each group will TAKE 2.0 mL of the liver puree and put it in a clean 100
mL beaker (labeled Liver Puree Solution) then ADD 78 mL distilled water to this beaker. This is
the liver puree solution you will be using for your trials!
PART A: Effect of Enzyme Concentration

1. Using a 10 mL graduated cylinder, add 8.0 mL of H2O2 to the 100 mL graduated cylinder.

2. Using a 10 mL graduated cylinder, measure 1.0 mL of the diluted liver puree. Add the liver puree to
the H2O2 in the 100 mL graduated cylinder.

3. Record the maximum height (in mL) of the foam produced. Be sure to subtract the volume of
solution at the bottom of the graduated cylinder.

4. Rinse the 100 mL graduated cylinder in the waste bin. Repeat steps 1, 2, and 3 using 2 mL of diluted
liver puree, then 4 mL, then 8 mL and 10 mL.

PART B: Effect of pH

1. Prepare the following mixtures in three separate test tubes:

Test Tube Mixture

A 5 mL diluted liver puree and 2 drops of 1.0 M HCl (pH = 1)
B 5 mL diluted liver puree and 2 drops H2O (pH = 7)
C 5 mL diluted liver puree and 2 drops of 1.0 M NaOH (pH = 14)

2. Add 8 mL of H2O2 to the 100 mL graduated cylinder. Add the contents of test tube A to the 100 mL
graduated cylinder. Observe and record the maximum height of foam produced.

3. Rinse the 100 mL graduated cylinder and empty the contents in the waste bin. Repeat steps 2 and 3
with test tubes B and C.

PART C: Effect of Temperature

Note you MUST record the ACTUAL temperature that you read on the thermometer just when
you are mixing the H2O2 and liver puree. If it says 22C, write it down as 22C and not 20C. The
temperatures below are suggested values only! Do not waste time waiting!

1. Add 8 mL of H2O2 to the 100 mL graduated cylinder. Add 5 mL of the liver puree to a test tube.
Immerse the test tube in an ice bath until the liver puree reaches a temperature of approximately
10C.

2. Pour the contents of the test tube into the 100 mL graduated cylinder. Observe and record the
maximum height of foam produced.

3. Repeat the above procedure with the liver puree at room temperature (approximately 20C).

4. Fill up a 500 mL beaker with tap water. Place this beaker on a hot plate and turn the hot plate on to
start a rolling water bath. This will be your water bath. Have three test tubes ready and put them
 aside. Add 5 mL of the liver puree to a test tube. Repeat this for the other two test tubes. Place the
three test tubes in the rolling water bath to warm them up.
5. Add 8 mL of H2O2 to a 100 mL graduated cylinder. Put aside.

6. When the temperature has reached approximately 40C, take ONE of the test tubes out of the beaker
using hot gloves or test tube tongs. Pout the contents (carefully!) of this test tube into the 100 mL
graduated you had set aside. Observe and record the maximum height of foam produced.

7. Rinse the 100 mL graduated cylinder and empty the contents in the waste bin.

8. Repeats steps 5, 6 and 7 for the other two test tubes (approximately 60C and 80C).

Observations:

Record your lab data in the spaces provided.

Part A Effect of Enzyme Concentration

Max. Height Change

Vol. of H2O2 Vol. of Liver Total Vol. (mL)
of Foam in Vol. Visual Observations
(mL) Puree (mL) (H2O2 + Puree)
(mL) (mL)

Notes:

Part B Effect of pH

Vol. of H2O2 Vol. of Liver Total Vol. (mL) Max. Height Change pH Visual
 of Foam in Vol.
(mL) Puree (mL) (H2O2 + Puree) Observations
(mL) (mL)

Notes:

Part C Effect of Temperature

Total Vol.
Max. Height Change
Vol. of H2O2 Vol. of Liver (mL) Temp Visual
of Foam in Vol.
(mL) Puree (mL) (H2O2 + (C) Observations
(mL) (mL)
Puree)

Notes:

Results:

You will create THREE graphs (one for each factor). The graphs should be formatted according to the
SBI4U lab write-up guidelines. It is recommended you use Microsoft Excel to create the graphs.

Discussion:

*** This is a FORMAL lab report. Follow the SBI4U Formal Lab Report Guidelines thoroughly ***

For the Discussion section of your formal lab report, you should have ALL components of the
Discussion section as requested in the lab report guidelines (re-state purpose of lab, state
observations/results, explain the results using course connections, account for errors, refer to hypotheses
and provide a general conclusion, etc).

Within the body of your Discussion section, include well-composed (and supported) answers (in
paragraphs) to the following questions for EACH factor that you tested.

Compare the data your group collected to your hypothesis for the factor. State whether your
hypothesis was correct. If not, why do you think this is? (consider sources of error in the design
or made during conducting the lab)

How does the factor affect the enzyme activity? Explain scientifically using course content.
What trends did you observe in your graphical results for the factor? How would you explain the
trends? Make specific reference to the data collected (and your graph) to help you explain.

What is the optimum value for this factor? (Do this ONLY for temperature and pH)

If you were to repeat this experiment, what would you change to improve it, and why? What
errors did you encounter? Why? What proposed solutions do you have in mind?

Evaluation Scheme:

Level
Code Expectation
Mark
Demonstrate scientific investigation skills (related to both inquiry and
A1 research) in the four areas of skills (initiating and planning, performing and
recording, analyzing and interpreting, and communicating).
Investigate the chemical structures, functions, and chemical properties of
B2 biological molecules involved in some common cellular processes and
biochemical reactions.
 I R 1- 1 1+ 2- 2 2+ 3- 3 3+ 4- 4 4+ 4++
A1. Demonstrate scientific investigation skills (related to both inquiry and research) in the four areas of skills (initiating and
planning, performing and recording, analysing and interpreting, and communicating).
Make informed educated hypotheses to
focus inquiries or research.
Select appropriate instruments and
materials, and identify appropriate
methods, techniques, and procedures.
Apply knowledge and understanding of
safe laboratory practices and procedures.
Using appropriate materials and
equipment safely, accurately, and
effectively, to collect observations and
data.
Compile accurate data from laboratory
and other sources, and organize and
record the data using tables. Limited Approaching Sufficient Insightful
Synthesize, analyze, interpret and
evaluate qualitative and/or quantitative
data to determine whether the evidence
supports or refutes the initial hypotheses;
identify sources of error; and suggest
improvements to the inquiry to reduce the
likelihood of error.
Draw conclusions based on inquiry
results and justify their conclusions with
reference to scientific knowledge.
Communicate ideas, results and
conclusions in writing using appropriate
language and a variety of formats.
I R 1- 1 1+ 2- 2 2+ 3- 3 3+ 4- 4 4+ 4++
B2. Investigate the chemical structures, functions, and chemical properties of biological molecules involved in some common
cellular processes and biochemical reactions
Use appropriate terminology related to
biochemistry.
Plan and conduct an investigation
related to a cellular process (factors that
Limited Approaching Sufficient Insightful
affect enzyme activity) using
appropriate laboratory equipment and
techniques, and report the results in an
appropriate format.