Department of Chemical Engineering

University of Wisconsin – Madison

CBE 424 – Operations and Process Laboratory

Informal 1
Hydrogen Peroxide Decomposition Using Bovine Catalase

Christian Fabian
Len Roche
Experiment Date:
07/11/13
Instructor:
Jiménez
Abstract

Hydrogen peroxide was allowed to decompose under the influence of

catalysts at various temperatures, pH values, and substrate concentrations.

Specifically, potassium iodine and bovine catalase were used to increase the rate

of decomposition to adequate levels. Furthermore, the decomposition was used to

characterize catalase under various conditions. The effects of temperature, pH,

and initial enzyme and substrate concentration on decomposition rates were

used to accomplish this. Enzyme performance was then modeled by Michaelis-

Menten kinetics. The 𝐾𝑚 for the decomposition was determined to be 0.1208 M,

while the 𝑉𝑚𝑎𝑥 found was 0.0097 M/min. Lastly, as hypothesized, catalase

activity is a maximum at bovine body temperature (~40 ºC) and blood pH of 7.

i
Table of Contents

Abstract i

Introduction and Theory 1

Procedure 2
Startup 2
Adjusting Temperature 3
Adjusting pH 4
Adjusting Enzyme Concentration 4

Results and Discussion 5

Conclusions 10

References 11

Nomenclature 12

Appendices 13
Supplementary Graphs and Figures 13
Original Data 13
Sample Calculations 16

ii
Introduction and Theory

Hydrogen peroxide undergoes decomposition to form oxygen and water

according to (1).

2𝐻2 𝑂2 → 2𝐻2 𝑂 + 𝑂2 (1)

this decomposition, however, is slow with typical concentrations (3-30 wt%)

found in household antiseptics and laboratory stock solutions. One way of

speeding the decomposition is to use a catalyst or enzyme. Both potassium iodide

and the enzyme catalase increase the rate of decomposition; hence these were

suitable for investigating the reaction order of (1).

A simple way of measuring the decomposition progress is to monitor the

pressure change △ 𝑝 over time 𝑡 in a closed vessel containing the solution of

hydrogen peroxide and a catalyst, or enzyme. This pressure change is then used

to determine the moles of oxygen 𝑛𝑂2 produced according to the Ideal Gas Law

(2). Pressure changes are expected to be small – deviations not far from

atmospheric pressure – so the ideal gas assumption should be valid. Finally,

using stoichiometry (3) and the volume of the solution 𝑉𝑠 one can find the

concentration of hydrogen peroxide 𝐶𝐻2 𝑂2 .

𝑝𝑉 = 𝑛𝑅𝑇 (2)

1 𝑛 𝑂2
𝐶𝐻2 𝑂2 = 2 (3)
𝑉𝑠

1
 The effects of concentration, temperature, and pH on the rate of

decomposition of 𝐻2 𝑂2 were further investigated using bovine catalase.

Michaelis-Menten kinetics (4) and Lineweaver-Burk plots (5) can be

implemented to characterize the enzyme and decomposition reaction

𝑉𝑚𝑎𝑥 [𝑆]
𝑉= (4)
𝐾𝑚 +[𝑆]

1 𝐾𝑚 1 1
=𝑉 +𝑉 (5)
𝑉 𝑚𝑎𝑥 [𝑆] 𝑚𝑎𝑥

where 𝑉 is the rate of decomposition, 𝐾𝑚 is the Michaelis-Menten constant, and

[𝑆] is 𝐻2 𝑂2 concentration in the solution.

Procedure

Startup

Determining the reaction order of hydrogen peroxide decomposition was

the primary goal of the investigation. Monitoring the decomposition using

pressure change requires that the volume of the vessel housing the hydrogen

peroxide solution be constant. Accomplishing this required a 125 ml filter flask

was connected to a monometer and closed off by a rubber stopper to contain the

oxygen gas produced. A stir bar was added to the flask to mix the reagents and

maintain a homogeneous mixture. Furthermore, a water bath was utilized to

keep the temperature of the flask constant – this is especially useful in the trials

2
using potassium iodide as the catalyst since the reaction releases heat. Figure 1

shows how the filter flask is connected to the monometer and covered by the

rubber stopper, as well as how the water bath is employed to keep a steady

temperature.

Figure 1. Apparatus for measuring the pressure changes resulting from

hydrogen peroxide decomposition.

Adequate amounts of both catalyst/enzyme and hydrogen peroxide had to

be determined to allow pressure changes within the range of the monometer

used in the experiments. Ultimately, a 1 ml aliquot of 3 wt% 𝐻2 𝑂2 mixed with 2

ml 𝐻2 𝑂 and approximately 0.1 g KI gave pressure changes of about 0.15-0.18 psi

(a suitable range for the monometer utilized). For the trials involving catalase, a

standard solution was prepared by dissolving 0.1 g bovine liver catalase in 50 ml

𝐻2 𝑂. In those runs 0.2 ml of the catalase solution was added to a 10 ml solution,

which contained 𝐻2 𝑂 and 𝐻2 𝑂2 at varying concentrations. Only in the trials

3
studying the effects of enzyme concentration did the added volume of catalase

vary.

Adjusting Temperature

Temperature effects were studied using the same apparatus as shown in

Figure 1, with the exception of an added temperature regulator that circulated

water in the bath. A temperature regulator was necessary because the changes

in temperature affect water vapor pressure, and it was essential that the flask

and solution were at the same temperature. Once in equilibrium, the monometer

was relieved of any pressure built up from the water vapor – this should be done

to avoid reading an excess pressure change not created by the oxygen.

Adjusting pH

Like many other enzymes, catalase functions are affected by pH. Testing

was done by measuring 0.2 ml of the catalase solution and addeding it to a 10 ml

solution, which contained 5 ml of 3 wt% 𝐻2 𝑂2 and 5 ml of a standard buffer

solution. The pH of the buffer solutions used were 1.0, 4.0, 7.0, and 10.0. Again,

the pressure changes caused by 𝐻2 𝑂2 decomposition and catalase were measures

by the apparatus described in the Startup section.

Adjusting Enzyme Concentration

Lastly, catalase concentration was varied to study its effect on 𝐻2 𝑂2

decomposition. The standard solution of catalase outlined in the Startup section

4
was used again, but this time the added volume was varied. Aliquots of 50 μl,

100 μl, 200 μl, 350 μl, and 400 μl from the catalase solution were added to a 10

ml solution, which contained 5 ml of 3 wt% 𝐻2 𝑂2 and 5 ml of 𝐻2 𝑂. Pressure

readings were taken again from flask-monometer apparatus.

Results and Discussion

Characterizing the decomposition required that the reaction order be

determined. For this, 𝐻2 𝑂2 was allowed to decompose under the catalytic

influence of potassium iodide. A plot of pressure change versus time revealed

that the pressure increased at a constant rate – this hinted that the

decomposition might be first order. To validate this hypothesis the pressure

changes first had to be correlated to 𝐻2 𝑂2 concentration changes over time.

Finally, integral analysis (assuming first order) on that data confirmed the

hypothesis. Figure 2 shows that the log of concentration of 𝐻2 𝑂2 is linear with

time.

5
 -1.22
0 50 100 150 200
-1.225

-1.23
ln(CH2O2) = -0.0002t - 1.2229
-1.235
ln(CH2O2)

1% H2O2 with KI catalyst

-1.24

-1.245 Integral analysis assuming

1st order
-1.25

-1.255

-1.26
Time (s)

Figure 2. Assuming a first order decomposition, the log of the concentration

(M) of hydrogen peroxide in the solution should be linear with time.

The same decomposition was allowed to run under the influence of a

bovine enzyme, catalase. This enzyme is common in numerous organisms, and

serves the purpose of protecting the cell from oxidative damage. 1 Just like other

enzymes, catalase activity is affected by its initial concentration, temperature,

pH, and substrate concentration. To study these effects, each one had to be

varied while the remaining factors were held constant.

As mentioned in Adjusting Enzyme Concentration, the volume of catalase

solution used was varied so as to span concentrations from 0.01 g/L to 0.08 g/L.

This was done to ensure observing a trend in the data, as well as to prevent

pressure changes outside the range of the monometer used. Figure 3 shows that

by increasing the initial concentration of catalase the rate of decomposition rises

exponentially. This trend can be attributed to the remarkable efficiency of

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catalase – a single molecule of the enzyme can decompose millions of 𝐻2 𝑂2 every

second. 2

0.016
decomposition (M/min)

0.014
0.012
Initial rate of

V = 0.0009e34.845E
0.01
0.008
0.006
0.004
0.002
0
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
Enzyme conc. (g/L)

Figure 3. Effect of catalase initial concentration on hydrogen peroxide

decomposition. A

Temperature is a factor that greatly influences the activity level of

catalase. From the experiments conducted, catalase activity improved by an

order of magnitude from 3.323× 10−3 M/min at 5.25 ºC to 1.173× 10−2 M/min at

41.3 ºC – this is a 253% increase in activity. The opposite is true for

temperatures above the denaturation temperature, somewhere above 41.3 ºC.

For instance, at 50 ºC the rate of 𝐻2 𝑂2 decomposition drops to 9.615× 10−3

M/min. Figure 4 presents this relationship between temperature and catalase

activity. One thing to note about the trend is that the maximum catalase activity

occurs around 40 ºC, which is in the range of cattle body temperature. 3 This is

7
expected because enzymes are most efficient when they are placed in

environments resembling their natural conditions.

0.014
decomposition (M/min)

0.012
Initial rate of

0.01

0.008

0.006

0.004

0.002

0
0 10 20 30 40 50 60 70
Temperature (ºC)

Figure 4. The rate of hydrogen peroxide decomposition increases with

temperature.

The pH effect was expected to have similar trends as those of

temperature. As with high and low temperatures, enzymes tend to be less

efficient at high and low pH. Again, this is due to denaturization of catalase at

extremely low pH. Based on what was learned from temperature effects, a

prediction was made that catalase should be the most efficient at a pH around 7.

This hypothesis was made because the pH of bovine blood is around 7. 3 Figure

5, which shows a maximum decomposition rate at around pH of 7, confirmed the

hypothesis. At pH of 1, catalase denatures and so it fails to catalyze the

decomposition of 𝐻2 𝑂2.

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 0.006

Rate of decomposition 0.005

0.004
(M/min)

0.003

0.002

0.001

0
0 2 4 6 8 10 12
pH

Figure 5. Effects of pH on the rate of decomposition of hydrogen peroxide. The

rate is at its maximum around pH of 7.

Lastly, initial substrate concentration effects were analyzed using

Michaelis-Menten kinetics. In order to successfully fit the data to that model,

substrate concentrations had to be varied without making them too

concentrated, i.e. carefully choosing concentrations that fell within the

Michaelis-Menten kinetics regime. For that reason, a range from 0.0265 M to

0.3528 M 𝐻2 𝑂2 was chosen. Figure 6 shows initial rates of decomposition

resulting from the variation of substrate concentration, as well as the fitted

Michaelis-Menten model. The data was also used to obtain values for 𝐾𝑚 and

𝑉𝑚𝑎𝑥 from a Lineweaver-Burk plot (Figure 7). The 𝐾𝑚 for the decomposition is

0.1208 M, while the 𝑉𝑚𝑎𝑥 is 0.0097 M/min.

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 0.009

Rate of decomposition
0.008
0.007
(M/min) 0.006
0.005
0.004
0.003
0.002
0.001
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Substrate Conc. (M)

Figure 6. Effects of initial substrate concentration on the rate of

decomposition. The line represents the fitted Michaelis-Menten model.

Conclusions

At low concentration, the rate of decomposition of hydrogen peroxide is

slow. Using potassium iodide as a catalyst, 𝐻2 𝑂2 decompostion was determined

to be first order with respect to its concentration. Furthermore, the results of

varying temperautre, pH, and initial enzyme and substrate concentrations

showed that 𝐻2 𝑂2 decomposition rates reach a maximum and deteriorate at

extremes, with the exception of initial enzyme concentration. Catalase function

was shown to be sensitive to temperatures and pH changes. It was also

confirmed that catalase activity was at its maximum when it was placed in

conditions that mimicked its natural environment, i.e. bovine body temperature

(~40 ºC) and blood pH of 7. 2

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 Further studies could improve on the methods of regulating the

temperature of the solution decomposing. This would also allow more data to be

collected, to the extent of being able to pin-point the denaturation temperature.

A similar approach can be taken for pH, where more data collection could reveal

exactly at what pH catalase activity ceases.

References

1. Chelikani P, Fita I, Loewen PC. (2004). Diversity of structures and properties

among catalases. Cell. Mol. Life Sci. 61 (2): 192–208.

2. Goodsell, David. (2004). Catalase. Molecule of the Month. RCSB Protein Data

Bank. Retrieved 7/15/13.

3. MacDonald, David. (1984). Mammals. Oxford: Equinox, 1984: 545.

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Nomenclature

△𝑝 pressure change (cm H2O, kPa)

𝑡 time (s)

𝑛𝑂2 moles of oxygen produced

𝑉𝑠 volume of the solution (L)

𝐶𝐻2 𝑂2 concentration of hydrogen peroxide (M)

𝑉 rate of decomposition (M/min)

𝑉𝑚𝑎𝑥 maximum rate of decomposition (M/min)

𝐾𝑚 Michaelis-Menten constant (M)

[𝑆] 𝐻2 𝑂2 concentration in the solution (M)

𝑅 gas constant (L-kPa/K-mol)

𝑇 temperature (ºC)

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Appendices

Supplementary Graphs and Figures

700

600

500 1/V = 12.478(1/S) + 103.328

1/V (min/M)

400

300

200

100

0
0 10 20 30 40
1/S (M-1)

Figure 7. Lineweaver-Burk plot used to find Vmax and Km.

Original Data

1% H2O2 with KI catalyst

Run 1 Run 2
Time (s) cm H2O Time (s) cm H2O
0 0 0 0
10 0.3 10 0.3
20 0.7 15 0.6
30 1.3 20 1
35 1.6 25 1.3
40 1.9 30 1.6
45 2.3 35 2.05
50 2.6 40 2.4
55 3 45 2.7
60 3.4 50 3.3
65 3.7 55 3.7
70 4.1 60 4
75 4.5 65 4.35

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 80 4.85 70 4.7
85 5.3 75 5.1
90 5.6 80 5.45
95 5.95 85 5.7
100 6.3 90 6.2
105 6.7 95 6.5
110 7 100 6.9
115 7.4 105 7.3
120 7.8 110 7.6
125 8.2 115 8.1
130 8.5 120 8.4
135 8.9 125 8.7
140 9.2 130 9.1
145 9.7 135 9.5
150 10.1 140 9.7
145 10.3
150 10.6

Adjusting pH
H2O2 (ml) 5 H2O2 (ml) 5 H2O2 (ml) 5 H2O2 (ml) 5
H2O2 (ml) 5 H2O2 (ml) 5 H2O2 (ml) 5 H2O2 (ml) 5
Cat. (μl) 50 Cat. (μl) 50 Cat. (μl) 50 Cat. (μl) 50
Temp (ºC) 21 Temp (ºC) 21 Temp (ºC) 21 Temp (ºC) 21
pH 1 pH 4 pH 7 pH 10
cm cm cm cm
Time (s) H2O Time (s) H2O Time (s) H2O Time (s) H2O
0 0 0 0 0 0 0 0
10 0 10 1 10 2.9 5 0.6
20 0 20 2.5 20 7.4 10 1.5
30 4 30 10.8 15 2.6
40 5.7 40 14.1 20 4.3
50 6.9 50 17.4 30 6.9
60 8.1 40 9.2
50 11.7
60 13.1

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