Investigation of the rate of decomposition of

hydrogen peroxide into oxygen and water in the

presence of catalase
Abstract

Enzymes are mostly biological proteins that are very prominent in living

organisms. They lower the activation energy that is required in chemical reactions

without actually participating and so they are known as a type of catalyst. Enzyme

catalysis is almost indispensable for many organic processes, where uncatalyzed

reactions would otherwise require high temperatures unsuitable for life. In this lab we

examined the rapid conversion of hydrogen peroxide into water and oxygen by the

enzyme catalase, which is prevalent in liver cells. We observed the gradual descent of the

enzyme catalysis rate due to a reduction in substrate concentration and increase in

product concentration over time. We saw the effect of external factors such as pH and

temperature on the function of the enzyme. Furthermore we had to assay for the base line

at the beginning of the experiment and exercise our titration techniques.

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 Introduction

Enzymes are mostly biological proteins that are very prominent in living

organisms. They lower the activation energy that is required in chemical reactions

without actually participating and so they are known as a type of catalyst. A properly

functioning enzyme (E) always has a particular three-dimensional shape with specific

active sites which enable it to bind to another substance, usually called a substrate (S) that

has a complementary shape and needs to be catalyzed. The interaction between an

enzyme and its substrate happens as follows: E + S → ES → E + P. Once the substrate

bonds with the enzyme, its shape will change and previous chemical bonds will break.

The resulting product (P) then separates from the substrate. Since the enzymes are never

used up, they are constantly reused in various important biological processes. If the active

sites of enzymes are adversely blocked or affected by foreign particles, then the enzymes

will denature and effectively cease to function.

There are many other environmental factors that could easily denature enzymes.

A salt concentration that is too high will interrupt regular interactions between charged

particles, and one that is too low will lead the charged enzyme side chains to attract each

other. In the solution where enzymes are usually dissolved, the pH, the scale of hydrogen

cation concentration for which 0 is highest and 14 lowest, is crucial. A high or low PH

has the potential to make the enzyme either gain or lose so many hydrogen ions that it

loses its distinctive shape and denatures. Temperature, the measure of average kinetic

movement caused by heat, is generally positively related to reaction rates. However, there

is a threshold where temperature is so high that the enzyme no longer can bind properly

with its substrate, and so reaction rates decrease from thereon. At very high temperatures

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such as boiling temperature, most enzymes are denatured. Particles that can affect the

enzyme consist of activators that increase the reaction rate and inhibitors that decrease

the reaction rate. Also there are noncompetitive inhibitors that bind away from the active

site and competitive inhibitors that bind with the active site1.

The enzyme that is being investigated is catalase. It is vital to aerobic metabolic

processes, and so is present in virtually all living organisms, including humans. For

example, when electrons are moved to reduce oxygen into water as part of respiration, an

extra electron can be acquired, creating hydrogen peroxide (H2O2), a powerful and

harmful oxidizing agent2. Hydrogen peroxide is also created frequently during the

production of ATP. Uncatalyzed H2O2 spontaneously decomposes at a minimal rate in 24

hours. Consequently, to prevent damage in human cells, hydrogen peroxide is transported

to an organelle called the peroxisome, where it decomposes swiftly into water and

oxygen in the presence of catalase3. Catalase is especially prevalent in liver cells.

Some inhibitors of catalase are cyanide, a potent poison, and copper sulphate, a

noncompetitive inhibitor. Real life applications of catalase include removal of H2O2

during cold sterilization and preventing oxidation in food wrappers.

Materials

Exercise 2A: 30mL of 1.5% (0.44M) H2O2, 2 50mL glass beakers, 100mL measuring

cylinder, beaker of fresh catalase solution in ice bucket, pipette, microwave, chicken

liver, tweezers, scissors

Exercise 2B, 2D: 70mL of 1.5% (0.44M) H2O2, 70mL of (1.0M) H2SO4, 1mL deionized

water, KMnO4 in a burette, clamp, stand, 2 50mL glass beakers, 100mL measuring

cylinder, goggles, beaker of fresh catalase solution in ice bucket, pipette, clock

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Procedure

Exercise 2A: Transfer 10mL of 1.5% H2O2 into a 50mL glass beaker and add 1mL of

catalase solution with a pipette. Observe changes in solution. Wash used glass beaker

thoroughly. Then put 5mL of catalase solution in the same 50mL glass beaker and place

in a microwave oven until it boils. Transfer 10mL of 1.5% H2O2 into another 50mL glass

beaker and add 1mL of the cooled, boiled catalase solution with a pipette. Observe

changes in solution. Pour away any remaining solution in the second glass beaker before

putting 10mL of 1.5% H2O2 in it. Cut 1cm3 of chicken liver with scissors and transfer it to

the beaker of H2O2 with tweezers. Observe any changes.

Exercise 2B: Wear goggles to protect eyes from H2SO4. Put 10mL of 1.5% H2O2 into a

clean glass beaker. Add 1mL of deionized water (instead of enzyme solution). Add 10mL

of (1.0M) H2SO4. Mix well. Remove and transfer a 5mL sample into another beaker and

assay for the amount of H2SO4. Use burette to add KMnO4 a drop at a time to the

solution, until a persistent brown color is obtained. Gently swirl the solution after adding

each drop. Record change in volume of KMnO4 in burette.

Exercise 2D: Wear goggles to protect eyes from H2SO4. Put 10mL of 1.5% H2O2 into a

clean glass beaker. Add 1mL of catalase extract. Swirl gently for either 10, 30 60, 90, 120

or 180 seconds. Then add 10mL of (1.0M) H2SO4. Remove and transfer a 5mL sample

into another beaker and assay for the amount of H2SO4. Use burette to add KMnO4 a drop

at a time to the solution, until a persistent brown color is obtained. Gently swirl the

solution after adding each drop. Record change in volume of KMnO4 in burette. Repeat

above steps until data for all 6 time intervals of catalyzed decomposition is obtained.

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Data

Exercise 2A
Table 1 records the observations made during various experiments.
Experiment Observations
Enzyme activity There was a fair amount of bubbling.
Effect of boiling There were no bubbles and no change in the solution.
Effect of chicken liver There was a fair amount of bubbling.

Exercise 2B
Mean Base Line: 3.10555555555556mL KMnO4

Exercise 2D
Table 2 describes the amount of substrate decomposed after several time intervals.
Time (seconds) 10 30 60 90 120 180
Base Line (mL) 3.11 3.11 3.11 3.11 3.11 3.11
Amount of KMnO4 consumed (mL) 2.05 0.6 2.8 0.8 0.375 0.05
Amount of H2O2 used (Base Line – 1.06 2.51 0.31 2.305556 2.73 3.055556
Amount of KMnO4 consumed)

Results

Exercise 2D
Table 3 displays the initial rate of reaction and the rates between each of the time points
Time Intervals 0-10 10-30 30-60 60-90 90-120 120-180
(seconds)
Rates (mL/s) 0.105556 0.125278 0.010185 0.076852 0.091019 0.050926
Graph 1 shows the amount of H2O2 decomposed by catalase at different time intervals

Amount of Hydrogen Peroxide Decomposed by Catalase over time

3.50
Amount of Hydrogen Peroxide

3.00

2.50

2.00
(mL)

y = 0.5661Ln(x) - 0.2975
1.50 2
R = 0.3076
1.00

0.50

0.00
0 30 60 90 120 150 180
Time (se conds)

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Conclusion

In Exercise 2A, the enzyme is catalase, the substrate is hydrogen peroxide and the

products are oxygen and water. The gas evolved from the reaction mixture is oxygen, and

it can be tested by placing a glowing splint over the bubbling mixture and see if the splint

glows brighter. After boiling the catalase solution, it no longer bubbled when it was

mixed with hydrogen peroxide, and thus no reaction was catalyzed to occur, since boiling

denatured the enzyme. Since the bubbling reaction mixture with the chicken liver

indicated that the liver contained catalase, there probably would be no reaction at all if

the liver was boiled prior its addition to hydrogen peroxide.

For Exercise 2B the amount of hydrogen peroxide originally present in the 1.5%

solution was determined to be 3.11mL.

In Exercise 2D the data obtained was plotted in graph 1, and the best fit line

drawn shows a steep slope at the beginning that gradually curves right. Along with table

3, it is evident that initially the rate of reaction was fastest, and it decreased as

concentration of hydrogen peroxide declined over time. The rate is lowest near the end at

the time point of 180 seconds, because concentration of catalase in the solution has also

declined due to the increased amount of water, a product of the reaction, that blocks the

substrate from reaching the active sites quickly. Sulphuric acid has an inhibiting effect on

the function of catalase, because it raises the pH of the solution dramatically, causing the

enzyme to acquire too many hydrogen ions, change shape and denature. Lowering the

temperature would reduce the rate of enzyme activity, and at extreme temperatures the

solution would freeze and the reaction would stop, since the average kinetic energy of

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molecules is lowered, lessening the chance of substrates to bump into and conjoin with

the active sites of enzymes.

A controlled experiment to test the effects of varying pH could be conducted by

placing acids and bases of different pH such as sulphuric acid and calcium chloride in

equal concentration and volumes in solution with the substrate, and then observe any

chemical changes after adding the enzyme.

In doing this lab, we learned the importance of establishing a base line as a form

of control for our experiment. We also realized that our titration techniques needed to be

refined, especially when we were studying changes by the drop. Finally we saw how

effective enzyme catalysis was in biology.

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Sources cited

9
1
About Catalase < http://www.ehow.com/about_5103294_catalase.html>
2
Catalase – An Extraordinary Enzyme <http://www.catalase.com>
3
What is the relationship between oxidase and catalase to aerobic respiration? <http://answers.yahoo.com/question/index?
qid=20080304005806AAqaPvS>