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 for the decomposition was determined to be 0.1208 M, while the 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.
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
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 one can find the concentration of hydrogen peroxide .
(2)
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 the rate of decomposition, is the Michaelis-Menten constant, and [ ] is 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
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% mixed with 2 ml 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
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% 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 is linear with time.
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
ln(CH2O2) = -0.0002t - 1.2229 -1.26 -1.255 -1.25 -1.245 -1.24 -1.235 -1.23 -1.225 -1.22 0 50 100 150 200 ln(C H 2O2 ) Time (s)
1% H2O2 with KI catalyst Integral analysis assuming 1st order
catalase – a single molecule of the enzyme can decompose millions of every second. 2
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 ºC to 1.173 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 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 ºC, which is in the range of cattle body temperature. 3 This is
V = 0.0009e34.845E 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 Initia l ra te of dec omposi tion (M /m in) Enzyme conc. (g/L)
expected because enzymes are most efficient when they are placed in environments resembling their natural conditions.
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 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0 10 20 30 40 50 60 70 Initia l ra te of de c omposi tion (M /m in) Temperature (ºC)
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 and
from a Lineweaver-Burk plot (Figure 7). The for the decomposition is
0.1208 M, while the is 0.0097 M/min.
0 0.001 0.002 0.003 0.004 0.005 0.006 0 2 4 6 8 10 12 Rate of de c omposi tion (M /m in) pH
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 ºC) and blood pH of 7. 2 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Rate of de c omposi tion (M /m in) Substrate Conc. (M)
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.
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)
Appendices
Supplementary Graphs and Figures
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 1/V = 12.478(1/S) + 103.328 0 100 200 300 400 500 600 700 0 10 20 30 40 1 /V (min/M) 1/S (M-1)
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 Time (s) Hcm 2O Time (s) cm H2O Time (s) cm H2O Time (s) cm 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
