Factors Affecting Enzyme Activity
Objectives:
 Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display data.
 Students know enzymes are proteins that catalyze biochemical reactions without altering the reaction equilibrium and the
activities of enzymes depend on the temperature, ionic conditions, and the pH of the surroundings.
Prelab Quesitons:
Research the following terms:
Enzymes, equilibrium, hydrogen peroxide, substrate, catalase, biological catalyst, activation energy, independent variable,
dependent variable
Write a hypothesis for each lab part. Explain the reason behind your hypothesis? (Do some research if you need to.)
Introduction:
Hydrogen peroxide (H2O2) is a poisonous byproduct of metabolism that can damage cells if it is not removed. Catalase is an enzyme
that speeds up the breakdown of hydrogen peroxide into water (H2O) and oxygen gas (O2).
2H2O2  2H2O + O2
REMEMBER: A CATALYST is a substance that lowers the activation energy required for a chemical reaction, and therefore
increases the rate of the reaction without being used up in the process. Catalase is
an enzyme, a biological catalyst, for which hydrogen peroxide is the substrate.
The testing system used in this lab consists of a small filter paper disc, which is coated with the enzyme and then placed into a beaker
of substrate (hydrogen peroxide). As the catalyst breaks down the hydrogen peroxide into water and oxygen gas, the bubbles of
oxygen cling to the filter and make it rise to the surface of the hydrogen peroxide. The time it takes for the filter to rise is an
indication of the rate of enzyme activity.
Rate of anything (like how fast a car is moving)is measured in units per time (like miles per hour (mi/hr)). After timing the time it
takes for the filter disc to rise you will convert this to a rate by dividing 1 by that number of seconds: rate = 1/sec). The enzyme has
been prepared for you as follows: 50g of peeled potato was mixed with 50 ml cold distilled water and crushed ice and homogenized in
a blender for 30 seconds. This extract was filtered through cheesecloth. ENZYME SHOULD BE KEPT ON ICE AT ALL TIMES!!
Steps common to all parts of the investigation:
1.
2.
3.
Always swirl the enzyme solution before dipping the disks. Using forceps, dip a filter paper disk into the appropriate
enzyme solution, then remove it and drain it quickly on a paper towel.
Place the disk into the beaker of hydrogen peroxide with forceps so that it sits on the bottom. When the disk touches the
substrate solution, begin timing, and record the number of seconds required for the disk to rise to the surface. Remove the
disk after it reaches the surface. If the disc still has not reached the surface after 120 seconds, remove the disc and write
down 120 seconds.
Perform three trials and record all your data. Calculate the average.
Part A. What is the effect of enzyme concentration on enzyme activity?
1.
2.
3.
Set up one 50 ml beaker containing 20 ml of 3% hydrogen peroxide.
Obtain at least 50 ml of enzyme in a 100ml beaker at use it to make the enzyme concentrations in the table below. Set up
each in a separate 50ml beaker. Label the beakers: a, b, c, d, e.
Take a paper disk, dip it solution a, drain the disk on a paper towel, and then place it on the bottom of the hydrogen peroxide
beaker. Measure the time it takes to rise to the top. Test all the solutions and then go back and do trial 2 of each, and then
again a third time.
How to mix solution of enzyme
a. 0% enzyme: add 0 enzyme stock to 20ml of water
b. 20% enzyme: add 4ml enzyme stock to 16ml water
c. 50% enzyme: add 10ml enzyme to 10ml water
d. 80% enzyme: add 16ml enzyme to 4ml water
e. 100% enzyme: add 20ml enzyme to 0ml water
trial 1 (sec.)
trail 2(sec.)
trial 3(sec.)
Average
time (sec.)
Rate
( 1/sec)
Part B. What is the effect of substrate concentration on an enzymatic reaction.
1.
2.
3.
Obtain 20 ml of enzyme in a 100 ml beaker.
Dilute the substrate (3 % hydrogen peroxide) as described below. Each dilution should be made in a separate 50ml beaker.
Label the beakers a, b, c, d, e.
Take a paper disc, dip it into the enzyme beaker, drain it on a paper towel and then place it on the bottom of beaker a. Record
the time it takes to rise to the surface. Repeat using beaker b, etc. Then repeat all the beakers again in trial 2, and again in
trial 3.
How to mix solution of substrate
trial 1 (sec.)
trail 2(sec.)
trial 3(sec.)
Average
time (sec.)
Rate
( 1/sec)
a. 0% substrate: add 0 H2O2 to 20ml of water
b. 0.38% substrate: add 2.5ml H2O2 w17.5ml water
c. 0.75% substrate: add 5ml H2O2to 15ml water
d. 1.5% substrate: add 10ml H2O2 to 10ml water
e. 3% substrate: add 20ml H2O2 to 0ml water
Part C. What is the effect of pH on enzyme activity?
1.
2.
3.
4.
Obtain 20 ml of 1% H2O2 in a 50ml beaker.
Obtain at least 25 ml of the enzyme in a 100 ml beaker and use it to make the solutions below.
Label 5 small beakers as follows: pH3, pH5, pH7, pH9, pH11 and dilute catalase into the appropriate beaker as directed
below in the table.
Dip a disc into the beaker at pH 3, drain it on a paper towel and then place it into the H2O2. Record the time it take to reach
the surface. Repeat the procedure for each pH. Then repeat all the pH beakers again in trial 2, and again in trial 3.
Different pH solutions to dip you disc in
trial 1 (sec.)
trail 2(sec.)
trial 3(sec.)
Average
time (sec.)
Rate
( 1/sec)
a. pH 3: 5ml catalase + 5ml pH3 buffer
b. pH 5: 5ml catalase + 5ml pH5 buffer
c. pH 7: 5ml catalase + 5ml pH7 buffer
d. pH 9: 5ml catalase + 5ml pH9 buffer
e. pH 11: 5ml catalase + 5ml pH11 buffer
Part D. What is the effect of temperature on enzyme activity?
1.
2.
3.
4.
5.
6.
7.
8.
9.
Obtain at least 20 ml of enzyme in a 100 ml beaker.
Place 5 ml of the enzyme in each of 4 test tubes.
Place 1 test tube in each of the four temperatures.
Place 20 ml 1% H2O2 in each of 2 50ml beakers.
Place 1 beaker in the 0oC bath and leave the other 3 beakers at room temperature. This is necessary because heat will destroy
the hydrogen peroxide.
Allow the enzyme and H2O2 to incubate at each temperature for about 5 minutes.
Transfer the enzyme that was at 0 degrees into a small container. Dip a paper disc into the cold enzyme, drain it on a paper
towel, and then place it into the cold H2O2. Record the time it take to rise to the surface.
Transfer the enzyme at room temperature into another container. Dip a paper disc into the enzyme, drain it on a paper towel,
and then place it into the room temp H2O2. Record the time it take to rise to the surface.
Repeat using the warmer enzymes and room temperature H2O2.
Different temperatures to test the reaction at
trial 1 (sec.)
trail 2(sec.)
trial 3(sec.)
Average
time (sec.)
Rate
( 1/sec)
a. 0 degrees (ice bath)
b. 22 degrees (room temperature)
c. 37 degrees (water bath)
d. 100 degrees (boiled enzyme)
Data:
For EACH variable, use the AVERAGE rates from the class data to construct a graph of the independent variable vs. the dependent
variable. This means you will have four graphs. USE GRAPH PAPER, A RULER FOR STRAIGHT LINES. MAKE YOUR
GRAPHS AT LEAST HALF A PAGE IN SIZE.
1. Enzyme concentration (x axis) vs. Rate (y axis)
2. Substrate concentration (x axis) vs. Rate (y axis)
3. pH (x axis) vs. Rate ( y axis)
4. Temperature (x axis) vs. Rate (y axis)
POST LAB QUESTIONS (Must answer all questions regardless of experiment your group conducted)
1.
2.
3.
4.
5.
What is the effect of enzyme concentration on enzyme activity? Explain how enzyme activity changes as enzyme
concentration decreases, and discuss why this occurs (on a molecular level).
What is the effect of substrate concentration on enzyme activity? How does enzyme activity change as substrate
concentration decreases? Explain your observations by discussing this reaction on a molecular level.
How does temperature affect the activity of catalase? Explain your observations by discussing the effect of temperature on
protein structure. Discuss both high and low temperature effects.
How does pH affect the activity of catalase? Consider both high and low pH, and explain your observations by discussing
the effect of pH on protein structure.
Ectothermic organisms have body temperatures that vary with the temperature of their surroundings. Discuss the effect this
variation might have on the functioning of enzymes in these organisms. Suggest some ways ectothermic organisms might
cope with this problem.
Conclusion: Did the results support or not support your hypothesis for each experiment?