Putmans Biol 160 Lab 5: Enzymology

LAB 5: Enzymology
Introduction
Every biochemical reaction that takes place within your body is controlled by a group of
proteins called enzymes. Enzymes are biological catalysts, acting to speed up chemical
reactions. The entire enzyme is called the holoenzyme. Holoenzymes consist of apoenzymes,
globular proteins that constitute most of the structure of an enzyme, and coenzymes or
cofactors, vitamin or mineral components that are largely responsible for the functioning of the
enzyme. It is necessary for you to eat a diet containing vitamins and minerals so that enzymes
can work properly!
Enzymes work by physically binding onto substrate molecule(s), forming an enzymesubstrate complex, then breaking the substrate molecule apart via hydrolysis or joining
substrates together via dehydration synthesis. Once enzymes are finished, they release the
finished product(s). This they can do many times because they do not chemically participate in
the reactions they catalyze.
Figure 5.1. Enzyme catalysis proceeds through the formation of an enzyme-substrate complex.
Top reaction is an enzyme-catalyzed hydrolysis, bottom a dehydration synthesis.

products

substrate
enzyme-substrate
complex

enzyme

H2O

substrates

product
enzyme

enzyme-substrate
complex
H2O

Enzymes must be a particular shape in order to fit with their substrate(s). Enzymes that
have had their shape changed irreversibly to the point they can no longer fit with their substrate,
hence have lost their catalytic ability, are said to be denatured. We know that pH, temperature,
salt concentrations and pressure affect the shape of proteins, so what would be the effect of these

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Putmans Biol 160 Lab 5: Enzymology

factors on the ability of enzymes to workon their reaction rates? Also, although shape is not a
question here, what about the concentration of enzymes and the concentration of substrate on
reaction rates?
A convenient enzyme to work with is catalase, one of the enzymes found within the cells
of both plants and animals; it is particularly easy to extract from animal liver, muscle tissue, and
potatoes. Catalase acts on the toxic substrate hydrogen peroxide, catalyzing its conversion to
oxygen gas and water:
2 H2O2

catalase

2 H2O + O2

Since peroxides are capable of destroying other molecules, including those of biological systems,
it is an advantage for cells to be able to remove peroxides from the body. And although hydrogen
peroxide does spontaneously decompose over time, it does so very slowly.

Laboratory Objectives
After mastery of this laboratory, including doing the assigned readings and required
laboratory work, the student should be able to:
1. Define and apply the terms
enzyme
cofactor
holoenzyme
enzyme-substrate complex
apoenzyme
denatured
coenzyme
2. Explain why changing the shape of an enzyme affects its reactivity and give the factors that
affect an enzymes shape.
3. Describe the source and effects of catalase.
4. Describe and demonstrate an experiment that would test the effects of enzyme concentration,
substrate concentration, pH and temperature on enzyme activity.
5. Graphically record and analyze enzyme data to determine optimal rates of enzyme activity at
various enzyme concentrations, substrate concentrations, pH values and temperatures.

Materials and Methods

Catalase Activity
1. Your instructor (or lab tech) will prepare for you a crude catalase extract by blending fresh
animal or plant tissue in DW then filtering it through cheesecloth. You need to obtain a test
tube of this filtrate for your experiments. Keep your test tube of catalase extract in ice until
you need to use it. (This retards decomposition of the catalase by other enzymes, including
those of bacteria.).
2. Each lab group should obtain a calibrated pipette to dispense the catalase extract, 9 test tubes
with test tube rack, a grease pencil and a 15 cm ruler. With the grease pencil, label the test

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tubes 1, 2, 3, 4, 5 and 6; test tubes 4, 5 and 6, and the other three well be using later.
3. Add the following to each tube then agitate and at 60 seconds record the results:
Tube 1: 1 mL catalase extract + 4 mL 1% hydrogen peroxide
Tube 2: 1 mL water + 4 mL 1% hydrogen peroxide
Tube 3: 1 mL catalase extract + 4 mL 2% sucrose solution
4. With the 15 cm ruler, measure the height of the column of bubbles (if any), in millimeters.
Record these data in your lab report. Rinse the test tubes for the next exercise.
Effects of Enzyme Concentration on Enzyme Catalysis Rates
1. First well make a dilution series of our catalase enzyme:
a. Into test tube 1, add 10 drops catalase + 10 drops DW. Agitate well. This is 50% catalase.
b. Into test tube 2, add 5 drops catalase + 15 drops DW. Agitate well. This is 25% catalase.
c. Into test tube 3, add 2 drops catalase + 18 drops DW. Agitate well. This is 10% catalase.
d. Into test tube 4, add 1 drop catalase + 19 drops DW. Agitate well. This is 5% catalase.
e. Finally, into test tube 5, add 20 drops (1 mL) DW. This is 0% catalase.
2. Add 1 mL of 1% hydrogen peroxide to each tube, agitate. At 60 seconds, with the 15 cm
ruler, record the height of the bubble column, in mm, in your lab report.
3. Rinse the test tubes for the next exercise.
4. Using these data, determine the optimal catalase extract concentration for the rest of this
labthe concentration that produces a good reaction without bubbling over the sides of the
test tube!
Effects of Substrate Concentration on Enzyme Catalysis Rates
1. As before, we need to make a dilution series, this time of our substrate, hydrogen peroxide:
a. Into test tube 1, place 20 drops (1 mL) of 3% hydrogen peroxide.
b. Into test tube 2, place 13 drops of 3% hydrogen peroxide + 7 drops DW. Agitate well.
This is 2% hydrogen peroxide.
c. Into test tube 3, place 10 drops of 3% hydrogen peroxide + 10 drops DW. Agitate well.
This is 1.5% hydrogen peroxide.
d. Into test tube 4, place 7 drops of 3% hydrogen peroxide + 13 drops DW. Agitate well.
This is 1% hydrogen peroxide.
e. Into test tube 5, place 3 drops of 3% hydrogen peroxide + 17 drops DW. Agitate well.
This is 0.5% hydrogen peroxide.
f. Finally, into test tube 6, place 20 drops (1 mL) of DW. This is 0% hydrogen peroxide
2. Using your seventh test tube, make 6 mL of your optimal catalase concentration. To do this,
simply multiple the number of drops used to make your optimal concentration by six.
3. Add 20 drops (1 mL) of your optimal catalase concentration to each tube, agitate and wait 60

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Putmans Biol 160 Lab 5: Enzymology

seconds before recording the results.
4. With a 15 cm ruler, measure the height of the column of bubbles, in millimeters. Record
these data in your lab report. Rinse the test tubes for the next exercise.
Effects of pH on Enzyme Catalysis Rates
1. In a clean 150 mL beaker, prepare 20 mL of your optimal hydrogen peroxide substrate
concentration by using the following table. What is left over from this exercise will be used
in the next, so dont throw it away!
substrate
concentration
needed
2.0 %
1.5 %
1.0 %
0.5 %

mL (drops) 3%
hydrogen peroxide
13.33 mL (267 drops)
10.00 mL (200 drops)
6.67 mL (133 drops)
3.33 mL (67 drops)

mL (drops) DW
6.67 mL (133 drops)
10.00 mL (200 drops)
13.33 mL (267 drops)
16.67 mL (333 drops)

2. You will be using 1.00 mL of your optimal catalase concentration, as determined above, for
this experiment, per test tube, but replacing the DW you used to make the optimal
concentration with pH buffers. To make 1.00 mL catalasebuffer solution, at your optimal
concentration, use the following table:
catalase
concentration
needed
50%
25%
10%
5%
0%

drops of pure catalase

extract
10 drops
5 drops
2 drops
1 drop
0 drop

drops of pH buffer
10 drops
15 drops
18 drops
19 drops
20 drops

3. Add the catalase-buffer solutions into the test tubes as follows:

a. Tube 1: catalase + pH 4 buffer
b. Tube 2: catalase + pH 6 buffer
c. Tube 3: catalase + pH 7 buffer
d. Tube 4: catalase + pH 8 buffer
e. Tube 5: catalase + pH 10 buffer
4. Add 20 drops (1 mL) of your optimal substrate concentration to each tube, agitate and wait
60 seconds before recording the results.
5. With a 15 cm ruler, measure the height of the column of bubbles, in millimeters. Record
these data in your lab report. Rinse the test tubes for the next exercise.

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6. Using these data, estimate the optimal pHthe pH at which you get the maximum amount of
gas production.
Effects of Temperature on Enzyme Catalysis Rates
1. Fill a 600 mL beaker 1/2 full of tap water. Set it on a hot plate and turn it on to full to boil the
water. Make sure you also have a beaker mitt and test tube clamp.
2. You should have enough optimal hydrogen peroxide concentration solution left from the
previous lab for this one (at least 4 mL); if not, prepare more as above!
3. Using a cleaned test tube and pipette, prepare 4 mL of your optimal catalase concentration at
your optimal pH, by multiplying by 4 the numbers of drops you used to make the optimal
concentration.
4. Prepare the test tubes as follows:
a. Test tubes 1-4, add 20 drops (1 mL) of optimal concentration, optimal pH catalase.
b. Test tubes 5-8, add 20 drops (1 mL) of optimal concentration hydrogen peroxide.
5. Place the test tubes in the following environments for at least 10 minutes each:
a. Test tubes 1 and 5 into the boiling water bath.
b. Test tubes 2 and 6 into incubator or warm water bath.
c. Test tubes 3 and 7 into refrigerator or ice bath.
d. Test tubes 4 and 8 on lab bench at room temperature.
6. In each of the four environments, note and record the temperature of the environment; DO
NOT insert the thermometers into your catalase or substrate solutions!!! The solutions will be
the same temperature as their environment after 10 minutes!
7. After at least 10 minutes, add the hydrogen peroxide to the catalase from the same
environment and agitate. (In other words, add test tube 5 to 1, 6 to 2, 7 to 3 and 8 to 4.) After
60 seconds, using the 15 cm ruler, note the height of the bubbles produced in each test tube,
in mm. Record these data in your lab report.
8. Rinse your test tubes and clean up as per instructions given in lab.

Make sure you cleanup your work station, clean all equipment you used
and put it back, and help in general to keep the lab clean and in order!

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Putmans Biol 160 Lab 5: Enzymology

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Putmans Biol 160 Lab 5: Enzymology

Biol 160
Lab 5: Enzymology
Prelab
(5 points)

Name: ___________________________________
Date: ________________ Lab Section: ________

~Complete this prelab before coming to lab; it is due at the beginning of lab!
1. __________________ speed up biochemical reactions within organisms.
2. __________________ is the technical term for an entire enzyme.
3. The protein structure of an enzyme is the __________________.
4. __________________ and __________________ are coenzymes/cofactors.
5. What is a substrate?

6. From the information in the introduction to the lab, describe how enzymes work!

7. What factors can affect enzyme activity by changing the shape of the apoenzyme?

8. What do we mean when we say that an enzyme has been denatured?

9. The enzyme ________________ converts H2O2 into H2O + O2.

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Putmans Biol 160 Lab 5: Enzymology

10. In our first exercise, Catalase Activity, which test tube (1, 2 or 3) do you predict will show
enzymatic activity?

11. Throughout these exercises, what product of catalase activity allows us to measure the rate of
catalase activity?

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Putmans Biol 160 Lab 5: Enzymology

Biol 160
Lab 5: Enzymology
Lab Report
(20 points)

Name: ___________________________________
Date: ________________ Lab Section: ________

Catalase Activity

Results
1. Reaction data for catalase activity.
Height of Bubble
Column (mm)

Tube Contents
1

1 mL catalase + 4 mL hydrogen peroxide

1 mL water + 4 mL hydrogen peroxide

1 mL catalase + 4 mL sucrose solution

Discussion
2. Which test tube (1, 2 or 3) was the positive control? ______ Negative control? ______
3. What is the function of having a positive and negative control in this experiment?

4. Which test tube (1, 2 or 3) showed enzymatic activity? _________ Why?

5. Did you get enzymatic activity in test tube 3? ________ Why or why not?

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Putmans Biol 160 Lab 5: Enzymology

Effects of Enzyme Concentration on Enzyme Catalysis Rates

Results
6. Reaction data of hydrogen peroxide + varying concentrations of catalase.
1.0 mL of 1% hydrogen peroxide + 1.0 mL of catalase
with the following concentrations:
50%
25%
10%
5%
0%
catalase
catalase
catalase
catalase
catalase
Height of
bubble column
(mm)

Analysis
7. Make a smooth-line graph of the data above with the height of the gas evolved (mm) in 60
seconds on the y axis and percent catalase extract on the x axis. Label each axis and the units
you use on your graph!

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Putmans Biol 160 Lab 5: Enzymology

Discussion
8. Given a particular concentration of substrate, does increasing the enzyme concentration
indefinitely increase the rate of product formation? Why or why not?

Effects of Substrate Concentration on Enzyme Catalysis Rates

Results
9. Data of enzyme reaction rates from 50% catalase extract plus 1.0 mL of hydrogen peroxide
substrate with varying concentrations.
1.00 mL of optimal catalase extract + 1.0 mL of hydrogen peroxide
substrate with the following concentrations:
3.0%
2.0%
1.5%
1.0%
0.5%
0%
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2
Height of
bubble column
(mm)

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Putmans Biol 160 Lab 5: Enzymology

Analysis
10. Make a smooth-line graph of the data above with the height of the gas evolved (mm) in 60
seconds on the y axis and percent hydrogen peroxide solution on the x axis. Label each axis
and the units you use on your graph!

Discussion
11. Given a particular enzyme concentration, does increasing the substrate concentration
indefinitely increase the rate of product formation? Why or why not?

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Putmans Biol 160 Lab 5: Enzymology

Effects of pH on Enzyme Catalysis Rates

Results
12. Data of enzyme reaction rates from optimal substrate concentrations and optimal catalase
concentrations at varying pH values.

pH
Height of
bubble column
(mm)

1.00 mL of buffered catalase extract at

optimal concentration + 1.00 mL of
optimal hydrogen peroxide substrate
concentration
4.0
6.0
7.0
8.0
10.0

Analysis
13. Graph the data above with the height of the gas evolved (mm) in 60 seconds on the y axis
and pH on the x axis. Label each axis and the units you use on your graph!

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Putmans Biol 160 Lab 5: Enzymology

Discussion
14. Based on your graphical analysis, what is the optimal pH for catalase activity?

15. What can you infer about the environment in which catalase is found from these data?

Effects of Temperature on Enzyme Catalysis Rates

Results
16. Data of enzyme reaction rates from optimal substrate concentrations and optimal catalase
concentrations at varying temperatures.
1.0 mL catalase extract at optimal
concentration and pH + 1.0 mL
hydrogen peroxide substrate at
optimal concentration:
Temperature of
catalase and
peroxide ( C)
Height of
bubble column
(mm)

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Putmans Biol 160 Lab 5: Enzymology

Analysis
17. Make a smooth-line graph of the data above with the height of the gas evolved (mm) in 60
seconds on the y axis and the temperature on the x axis. Label each axis and the units you use
on your graph!

Discussion
18. Based on your graphical analysis, what is the optimal temperature for catalase activity?

19. What can you infer about the environment in which these enzymes are found from these
data?

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Putmans Biol 160 Lab 5: Enzymology

Post Lab Questions

20. From an enzymatic perspective, why are homeothermic animals, animals with a constant
body temperature such as mammals and birds, more active than poikilothermic animals such
as snakes and lizards?

21. From an enzymatic perspective, why do animals die when we bring them up from the deep
sea?

22. Again, from an enzymatic perspective, why is it vital for blood to have a constant pH?

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