Department of Chemical EngineeringUniversity of Wisconsin – Madison

CBE 424 – Operations and Process LaboratoryInformal 1

Hydrogen Peroxide Decomposition Using Bovine Catalase

Christian FabianLen 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 V max 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).

2H 2O2→2H 2O+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 nO 2

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 V s one can find the concentration of

hydrogen peroxide CH 2O2.

pV=nRT (2)

1

CH 2O2=12

nO2V s

(3)

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

decomposition of H 2O2 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

V=V max [S ]Km+ [S ]

(4)

1V

=K m

V max

1[S ]

+ 1V max

(5)

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

constant, and [S ] is H 2O2 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

2

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 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%

H 2O2 mixed with 2 ml H 2O 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

3

prepared by dissolving 0.1 g bovine liver catalase in 50 ml H 2O. In those

runs 0.2 ml of the catalase solution was added to a 10 ml solution, which

contained H 2O and H 2O2 at varying concentrations. Only in the trials

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% H 2O2 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 H 2O2

decomposition and catalase were measures by the apparatus described in

the Startup section.

4

Adjusting Enzyme Concentration

Lastly, catalase concentration was varied to study its effect on H 2O2

decomposition. The standard solution of catalase outlined in the Startup

section 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%

H 2O2 and 5 ml of H 2O. Pressure readings were taken again from flask-

monometer apparatus.

Results and Discussion

Characterizing the decomposition required that the reaction order

be determined. For this, H 2O2 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 H 2O2 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 H 2O2 is linear with time.

5

0 20 40 60 80 100 120 140 160

-1.26

-1.25

-1.24

-1.23

-1.22

-1.21

-1.2

f(x) = − 0.000214928741872406 x − 1.22287272100055

1% H2O2 with KI catalystIntegral analysis assuming 1st order

Time (s)

ln(C

H2

O2

)

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

6

catalase the rate of decomposition rises exponentially. This trend can be

attributed to the remarkable efficiency of catalase – a single molecule of

the enzyme can decompose millions of H 2O2 every second. 2

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.090

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

f(x) = 0.000924126737539129 exp( 34.8454252398597 x )

Enzyme conc. (g/L)

Init

ial rate o

f decom

posit

ion

(M/m

in)

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 H 2O2 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

7

range of cattle body temperature. 3 This is expected because enzymes are

most efficient when they are placed in environments resembling their

natural conditions.

0 10 20 30 40 50 60 700

0.002

0.004

0.006

0.008

0.01

0.012

0.014

Temperature (ºC)

Init

ial ra

te o

f decom

posit

ion

(M/m

in)

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

8

1, catalase denatures and so it fails to catalyze the decomposition of H 2O2

.

0 2 4 6 8 10 120

0.001

0.002

0.003

0.004

0.005

0.006

pH

Rate

of

decom

posit

ion

(M/m

in)

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 H 2O2 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 V max from a Lineweaver-Burk plot (Figure 7). The

Km for the decomposition is 0.1208 M, while the V max is 0.0097 M/min.

9

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

0.0010.0020.0030.0040.0050.0060.0070.0080.009

Substrate Conc. (M)

Rate

of

deco

mpo-

sit

ion (

M/m

in)

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, H 2O2 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 H 2O2 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

10

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)

nO 2 moles of oxygen produced

V s volume of the solution (L)

CH 2O2 concentration of hydrogen peroxide (M)

V rate of decomposition (M/min)

V max maximum rate of decomposition (M/min)

Km Michaelis-Menten constant (M)

[S ] H 2O2 concentration in the solution (M)

R gas constant (L-kPa/K-mol)

T temperature (ºC)

12

Appendices

Supplementary Graphs and Figures

0 5 10 15 20 25 30 35 400

100

200

300

400

500

600

700

f(x) = 12.478296405284 x + 103.328281873752

1/S (M-1)

1/V

(m

in/M

)

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

Original Data

1% H2O2 with KI catalystRun 1 Run 2

Time (s) cm H2O Time (s) cm H2O0 0 0 0

10 0.3 10 0.320 0.7 15 0.630 1.3 20 135 1.6 25 1.340 1.9 30 1.645 2.3 35 2.0550 2.6 40 2.455 3 45 2.760 3.4 50 3.365 3.7 55 3.770 4.1 60 475 4.5 65 4.35

13

80 4.85 70 4.785 5.3 75 5.190 5.6 80 5.4595 5.95 85 5.7

100 6.3 90 6.2105 6.7 95 6.5110 7 100 6.9115 7.4 105 7.3120 7.8 110 7.6125 8.2 115 8.1130 8.5 120 8.4135 8.9 125 8.7140 9.2 130 9.1145 9.7 135 9.5150 10.1 140 9.7

145 10.3150 10.6

Adjusting pHH2O2 (ml) 5 H2O2 (ml) 5 H2O2 (ml) 5 H2O2 (ml) 5H2O2 (ml) 5 H2O2 (ml) 5 H2O2 (ml) 5 H2O2 (ml) 5Cat. (μl) 50 Cat. (μl) 50 Cat. (μl) 50 Cat. (μl) 50Temp (ºC) 21

Temp (ºC) 21

Temp (ºC) 21

Temp (ºC) 21

pH 1 pH 4 pH 7 pH 10

Time (s)cm H2O Time (s)

cm H2O Time (s)

cm H2O Time (s)

cm H2O

0 0 0 0 0 0 0 010 0 10 1 10 2.9 5 0.620 0 20 2.5 20 7.4 10 1.5

30 4 30 10.8 15 2.640 5.7 40 14.1 20 4.350 6.9 50 17.4 30 6.960 8.1 40 9.2

50 11.760 13.1

14