Name: ________________________________________________ Date: ___________________
AP Biology
Enzyme Catalysis Inquiry Lab
Background:
In general, enzymes are proteins produced by living cells; they act as catalysts in biochemical
reactions. A catalyst affects the rate of a chemical reaction. One consequence of enzyme activity is
that cells can carry out complex chemical activities at relatively low temperatures.
In an enzyme-catalyzed reaction, the substance to be acted upon (the substrate = S) binds
reversibly to the active site of the enzyme (E). One result of this temporary union is a reduction in
the energy required to activate the reaction of the substrate molecule so that the products (P) of
the reaction are formed.
E + S  ES  E + P
Note that the enzyme is not changed in the reaction and can be recycled to break down additional
substrate molecules. Each enzyme is specific for a particular reaction because its amino acid
sequence is unique and causes it to have a unique three-dimensional structure. The active site is
the portion of the enzyme that interacts with the substrate, so that any substance that blocks or
changes the shape of the active site affects the activity of the enzyme.
Hydrogen peroxide (H2O2) is a common but poisonous by-product of cellular metabolism, but H2O2
does not accumulate in cells because it is decomposed to water and oxygen gas. The
decomposition of the hydrogen peroxide is mediated by catalase, an enzyme present in most cells.
The balanced equation for the reaction is 2H2O2 -----> 2H2O + O2. One molecule of catalase can
catalyze the decomposition of approximately 4 x 107 molecules H2O2 per second!
The enzyme used in this lab – catalase – has four polypeptide chains, each composed of more than
500 amino acids. This enzyme is found throughout aerobic organisms. One function of catalase
within cells is to prevent the accumulation of toxic levels of hydrogen peroxide (formed as a byproduct of metabolic processes). Catalase may also take part in some of the many oxidation
reactions that occur in all cells. The primary reaction catalyzed by catalase is the decomposition of
H2O2 to form water and oxygen:
2H2O2  2H2O + O2 (gas)
In the absence of catalase, this reaction occurs spontaneously, but very slowly. Catalase speeds up
the reaction considerably. In this experiment, the rate of reaction will be indirectly measured. The
assay system used in this lab consists of a filter paper disk that is coated with the enzyme and then
dropped into a cup of substrate (hydrogen peroxide). As the catalyst breaks down the hydrogen
peroxide into water and oxygen gas, the bubbles of oxygen collect underneath the filter paper disk
and make it rise to the surface of the hydrogen peroxide. The time it takes for the filter paper disk
to rise (from the bottom of the cup) is an indication of the rate of enzyme activity.
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RATE ENZYME ACTIVITY = DISTANCE DEPTH OF HYDROGEN PEROXIDE IN MM/TIME IN SEC
We will assume that each filter disk is coated with the same amount of catalase (except in the
investigation of the effect of enzyme concentration of enzyme activity).
Safety:
You will be using glass, hot water, acids, and bases. Use caution and wear goggles.
Directions:
You will be working with your lab partner to design an experiment to test the activity of the
enzyme catalase. At this point, you should be well-versed in the following:
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The general functions and activities of enzymes
The relationship between the structure and function of enzymes
The concept of initial reaction rates of enzymes
In general, how environmental changes (changes in temperature, pH, enzyme
concentration, salt concentration, and substrate concentration) might affect the reaction
rates and efficiency of enzyme-catalyzed reactions.
Basic Catalase Assay Procedure: (use this as a reference for how to set up your basic protocol):
1. Prepare catalase using mortar and pestle, strainer and distilled water
2. Pour 20 mL H2O2 into a 25 mL graduated cylinder (or a tall cup with mm markings)
3. Pick up 1 filter paper disc with forceps
4. Soak the filter paper disc in catalase solution for 5 seconds and then air dry
5. Drop disc into graduated cylinder and start stopwatch as soon as the disc hits the surface of
the H2O2
6. Stop the stop watch when the disc reaches the top of the liquid in the graduated cylinder
7. Measure the total distance in mm the disc travels
Note: use the mL markings as if they were mm
Note: for some of the trials, the disc may not reach the bottom of the cylinder!
Note: total distance is distance the disc travels down and back up!
8. Record the time in seconds
9. Calculate rate as mm/s
(Note: the disc traveled 20 mL down and then 20 mL back up to the surface!)
10. Repeat 3—5 times and calculate average rate
You will need to work with your partner to prepare and run a detailed experiment testing the
effect of something on enzyme activity. You will need to decide what variable you will be
manipulating (such as temperature, pH, enzyme concentration, substrate concentration, salinity,
or another that requires prior teacher approval).
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Basic Lab Report Format:
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Problem (in the form of a question)
Hypothesis (in the form of a statement)
Dependent variable (the data you will collect)
Independent variable (what you will be manipulating)
Standardized variables (list at least 4 factors that must be kept equal in all treatments)
Levels of treatment (values set for the independent variable)
Control treatment (independent variable either eliminated or set to standard value)
Materials list
Protocol (be extremely specific, so another could read your report and completely replicate
the experiment)
Observations, Data Table(s), & Graphs
Analysis
Conclusion
Questions to ponder
o Construct 5 questions that you would ask someone who repeated your lab protocol
to see if they understand what the lab tested and the results that were achieved.
Then prepare answers to those questions.
Experimental Research Proposal Form attached (each person needs their own)
The final lab report must be typed. Laptops will be available in the classroom for this purpose.
The lab groups are partner teams. You may not perform the same experiment as the other team
at your table.
Anticipated Plan for Completion of Lab:
Day One (two periods)
Brainstorm ideas for lab
Complete Experimental
Research Proposal Form
Specifically outline protocol
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Day Two (one period)
Collect data
Day Three (one period)
Analyze data
Prepare lab report
EXPERIMENT RESEARCH PROPOSAL FORM
Teacher Approval ________________________________________________
1. Title ______________________________________________________________________
2. Team Members _____________________________________________________________
3. Research Relationship (What is affecting what?) ___________________________________
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4. Research Predication (What results do you expect?) ________________________________
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5. Hypothesis (Be specific. Include your variable, using an ”if…then…”format) _____________
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6. Experimental Design (Briefly describe the protocol) ________________________________
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7. Independent variable ________________________________________________________
8. Dependent variable __________________________________________________________
9. Standardized variables _______________________________________________________
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10. Levels of treatment (how will you vary the variable?) _______________________________
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11. Title of Data Table & Graph ____________________________________________________
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12. Proposed Plan to Analyze Data (In what way will you compare these numbers after you
collect them?) _____________________________________________________________
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13. Explain what results you would expect to see to support your hypothesis
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