Tiemann
BIOLOGY LAB: The Effect of Concentration on Enzyme Activity
PRE-LAB:
1. Define: enzyme, substrate, catalase, activation energy
2. Write the formula for calculating the reaction rate.
3. Draw the lab set-up.
Label: reaction chamber, stopper, pipette, graduated cylinder, discs, H2O2, container of water
BACKGROUND INFORMATION: (Summarize the information below in your own words.)
"Enzymes are proteins that act as catalysts that affect the rate of biochemical reactions. In an enzyme-catalyzed
reaction, the substance to be acted upon, the substrate (S), binds 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. 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
affect the activity of the enzyme. Changes in the environment also affect the function of enzymes." (Advanced
Placement Program Biology Lab Manual for Students, copyright 2001 by College Entrance Examination Board, p. 19)
The reaction may be summarized as: E + S → ES → E + P
Enzymes catalyze almost all chemical reactions that occur in living things. In cells, hydrogen peroxide (H 2O2) is
formed as a by-product of chemical reactions. One function of the enzyme catalase is to prevent the accumulation of
toxic levels of hydrogen peroxide, which may harm the organism. Catalase breaks down hydrogen peroxide into
water and oxygen gas by the reaction:
catalase
2H2O2
→
2H2O + O2
PURPOSE: How does enzyme concentration affect reaction rate?
HYPOTHESIS: Write an “if-then” statement expressing your expectations of the results as it relates to
the purpose of this lab.
MATERIALS: 3% hydrogen peroxide solution, 2-50mL beakers or clear plastic cups, 10-mL syringe,
yeast catalase solution, filter paper and hole punch, forceps, reaction chamber, one-hole rubber stopper
with plastic pipette, container of water, 50-mL graduated cylinder
PROCEDURE:
1. Place a small amount of yeast catalase in a small beaker or cup.
2. Punch out 10 discs from filter paper, place into the catalase solution, and allow to become
saturated.
3. Using forceps, remove one disc from the catalase solution and touch off any extra catalase
on the edge of your beaker. It should be saturated but not dripping. Place it on one interior
wall of your reaction chamber near the top.
4. Using a syringe, carefully measure out 10 mL of 3% hydrogen peroxide. While the reaction
chamber is up-right, add the hydrogen peroxide to the chamber.
Do not allow the H2O2 to make contact with the catalase!
5. Seal the chamber with a rubber stopper, which has a plastic pipette inserted into it. Gently
lay the reaction chamber in a container of water on its side with the disc of catalase facing up.
6. Fill a 50 mL graduated cylinder with water, cover the top, and invert the cylinder into your
container of water, keeping the mouth underwater at all times. Remove the cover. You should
have no air bubbles in your graduated cylinder. (This may require practice!)
7. Hold the graduated cylinder over the end of the pipette extending out of the reaction chamber.
8. Turn the reaction chamber 180 degrees so that the H2O2 comes into contact with the catalase.
9. Measure the gas levels in the graduated cylinder at 30-second intervals for 5 minutes.
Record this data in the data table.
10. Repeat steps 1-9 using 2 discs, 3 discs, and, finally, 4 discs. NOTE: If your graduated cylinder
fills up, have a second cylinder ready to trade places with the first one and continue your readings.
RESULTS:
Design a data table for your results
1. Write at least two observations about the experiment you performed.
2. Record the experimental results in a data table.
3. Using graph paper, prepare a line graph of the data collected for 1, 2, 3, and 4 discs. Plot the time on
the X-axis, and the amount of oxygen produced on the Y-axis. Label each axis with a proper title and
units. Give the graph a descriptive title. Include a brief description (not an explanation) of the graph on
the right-hand side.
CALCULATIONS: To determine a reaction rate, pick two points on the graph. Divide the difference in the
amount of product formed between these two points by the difference in time between them. Calculate the
reaction rate (mL O2/sec) using the following formula:
rate = Δy
Δx
SOURCE OF ERROR: Identify and explain at least two probable sources of error, weaknesses, and/or
suggestions for improvement in your experiment.
ANALYSIS QUESTIONS: (Answer in complete sentences so that the question is stated in the answer.)
1. What is the independent variable in this experiment?
2. What is the dependent variable in this experiment?
3. What type of biomolecule is an enzyme?
4. What is the enzyme in this reaction?
5. What is the substrate in this reaction?
6. a. How do you know that the gas evolved is oxygen?
b. Explain how you can experimentally test for the presence of oxygen.
7. What determines the 3-dimensional shape of a protein?
8. Predict the effect that lowering temperature would have on the rate of enzyme activity. Explain.
CONCLUSION: Using the third person objective-tense, explain the results of the investigation. Relate the
results to your hypothesis and to biochemistry.