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: Jimnez

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 MichaelisMenten kinetics. The while the for the decomposition was determined to be 0.1208 M,

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

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

Table of Contents Abstract Introduction and Theory Procedure i 1 2

Startup Adjusting Temperature Adjusting pH Adjusting Enzyme Concentration

Results and Discussion Conclusions References Nomenclature Appendices

2 3 4 4
5 10 11 12 13

Supplementary Graphs and Figures Original Data Sample Calculations

13 13 16

ii

Introduction and Theory Hydrogen peroxide undergoes decomposition to form oxygen and water according to (1). (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 over time 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 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 concentration of hydrogen peroxide . (2) (3) one can find the

The effects of concentration, temperature, and pH on the rate of decomposition of 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
[ ] [ ]

(4)

[ ]

(5)

where [ ] is

is the rate of decomposition, concentration in the solution.

is the Michaelis-Menten constant, and

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

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% ml mixed with 2

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 . In those runs 0.2 ml of the catalase solution was added to a 10 ml solution, which contained and 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% 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 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 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 l, 100 l, 200 l, 350 l, and 400 l from the catalase solution were added to a 10 ml solution, which contained 5 ml of 3 wt% and 5 ml of . Pressure

readings were taken again from flask-monometer apparatus.

Results and Discussion Characterizing the decomposition required that the reaction order be determined. For this, 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 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 time. is linear with

-1.22 -1.225 -1.23 ln(CH2O2) -1.235 -1.24 -1.245 -1.25 -1.255 -1.26 Time (s) Integral analysis assuming 1st order 0 50 100 150 200

ln(CH2O2) = -0.0002t - 1.2229

1% H2O2 with KI catalyst

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

catalase a single molecule of the enzyme can decompose millions of second. 2

every

0.016

Initial rate of decomposition (M/min)

0.014 0.012 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

V = 0.0009e34.845E

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 M/min at 5.25 C to 1.173 M/min at

41.3 C this is a 253% increase in activity. The opposite is true for temperatures above the denaturation temperature, somewhere above 41.3 C. For instance, at 50 C the rate of decomposition drops to 9.615

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 C, which is in the range of cattle body temperature. 3 This is

expected because enzymes are most efficient when they are placed in environments resembling their natural conditions.

Initial rate of decomposition (M/min)

0.014 0.012 0.01 0.008 0.006 0.004 0.002 0 0 10 20 30 40 50 60 70

Temperature (C)

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 .

0.006

Rate of decomposition (M/min)

0.005 0.004 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 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 from a Lineweaver-Burk plot (Figure 7). The 0.1208 M, while the is 0.0097 M/min. and

for the decomposition is

Rate of decomposition (M/min)

0.009 0.008 0.007 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, 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 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 C) 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): 192208.

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 pressure change (cm H2O, kPa) time (s) moles of oxygen produced volume of the solution (L) concentration of hydrogen peroxide (M) rate of decomposition (M/min) maximum rate of decomposition (M/min) Michaelis-Menten constant (M) [ ] concentration in the solution (M) gas constant (L-kPa/K-mol) temperature (C)

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Appendices

Supplementary Graphs and Figures

700 600

1/V (min/M)

500 400 300 200 100 0 0

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

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 13

80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

4.85 5.3 5.6 5.95 6.3 6.7 7 7.4 7.8 8.2 8.5 8.9 9.2 9.7 10.1

70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

4.7 5.1 5.45 5.7 6.2 6.5 6.9 7.3 7.6 8.1 8.4 8.7 9.1 9.5 9.7 10.3 10.6

H2O2 (ml) H2O2 (ml) Cat. (l) Temp (C) pH Time (s) 0 10 20

5 5 50 21 1 cm H2O 0 0 0

Adjusting pH H2O2 (ml) 5 H2O2 (ml) H2O2 (ml) 5 H2O2 (ml) Cat. (l) 50 Cat. (l) Temp (C) 21 Temp (C) pH 4 pH Time (s) 0 10 20 30 40 50 60 cm H2O 0 1 2.5 4 5.7 6.9 8.1 Time (s) 0 10 20 30 40 50

5 5 50 21 7 cm H2O 0 2.9 7.4 10.8 14.1 17.4

H2O2 (ml) H2O2 (ml) Cat. (l) Temp (C) pH Time (s) 0 5 10 15 20 30 40 50 60

5 5 50 21 10 cm H2O 0 0.6 1.5 2.6 4.3 6.9 9.2 11.7 13.1

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