Catalase Activity with Hydrogen Peroxide

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In the case that you unfortunate soul have to do this lengthy lab as well, I have here mine so you can take a gander and use it as a reference for your own write up!

 

Abstract

In this set of experiments the activity of the enzyme catalase as it reacts with hydrogen peroxide was observed by measuring oxygen molecules released as catalase decomposes hydrogen peroxide into molecular oxygen and water. In three experiments with varying concentrations of the enzyme, different temperatures and pH levels the rate of reaction was measured. Catalase breaks down hydrogen peroxide more efficiently at condition closest to the environment it is created to function in, the compartments of the body it is found in. Higher concentrations of the enzyme, temperatures closer to mammalian body temperature and pH levels closer to pH 7 yield the highest rates of reaction.

 

Introduction

Organisms use enzymes to convert hydrogen peroxide into water and molecular oxygen. Acting as a catalyst Catalase found in animals and protists, and peroxidase found in plants, speed up the chemical reaction making the substance into to 2H2O+ O2 from the toxic hydrogen peroxide. (1) Enzymes are very efficient and can do these reactions thousands of times a second as long as the ph and temperature are preferred to the enzyme (2). In this lab we will be using an O2, or oxygen sensor. Since oxygen is a product of the reaction, the concentration of the molecular oxygen will give an idea for which concentrations of the enzyme, pH of the solution, and temperature are most efficient at carrying out this reaction.  Using the O2 gas sensor, the oxygen levels as catalase reacts and at various concentrations will be measured. The production of oxygen as hydrogen peroxide is destroyed at different temperatures will be observed. Oxygen as hydrogen peroxide is decomposed at different pH values will be recorded.

It was hypothesized that the pH closer to 7 will yield higher amounts of oxygen being released. This is thought because the catalase is the most efficient at pH closer to body pH in compartments where catalase functions, which is 7.4. If the catalase is more efficient, then the hydrogen peroxide molecule will be broken down into two molecules of water and molecular oxygen at a higher rate. At a lower and a higher pH than 7-7.4, the enzyme will denature. Denaturation happens when the enzyme protein loses its shape and loses its function. The body pH is what the enzyme is constructed to function in.
It was hypothesized that the higher concentration of the enzyme catalase will yield higher rates of hydrogen peroxide decomposition because there will be more enzymes to break down the hydrogen peroxide molecule within the solution. More enzymes breaking down the hydrogen peroxide will yield more oxygen for the sensor, there for 3mL  will be the highest percent per second. It will reach saturation, the rate will not change: because oxygen will be determined based on the concentration that increases at the same rate.

The enzyme should be at its highest closer to body temperature. In this case, it is believed that 30 to 35 degrees will give higher molecular oxygen levels than at 0 to 5 degrees C, 20 to 25 degrees C and 50 to 55 degrees C. It is hypothesized that since the enzyme catalase was created to work in a mammalian body, this would be the case. It’s also believed that the enzyme will denature at a higher temperature because it will break down the enzyme and will no longer function.

 

Materials and Methods

Goggles and gloves were put on before the lab started. O2 Gas Sensor was connected to Lab Quest on the computer via a USB cord. The O2 sensor was set up manually: on the screen labeled Meter, Rate was tapped on. The data collection rate was changed to 0.2  samples per second and the data collection length to 180 seconds.

After the O2 sensor was set up on the Lab Quest program, Experiment 1 began in order to test different concentrations of enzymes’ effects on decomposing hydrogen peroxide. Three test tubes were labeled 1 2 & 3, each test tube was filled with 5 ml of 3% hydrogen peroxide and 5 mL of water. Next, the enzyme catalyzed reaction was initiated by these following steps; a clean dropper was filled with 5 drops of enzyme suspension and added to test tube labeled 1. The opening was covered with a finger and inverted two times gently. The content of tube one war poured into a clean 250 ml Nalgene bottle and the O2 sensor was placed on top of the bottle. The sensor was pressed gently into the opening and after 30 seconds, data collection started on the Lab Quest program. Data was collected and a graph for oxygen gas versus time was displayed.

Linear regression was performed to calculate the rate of reaction by first choosing curve fit from the Analyze Menu in Lab Quest. Linear was selected for the fit equation and the linear regression statistics for these two data columns were displayed for the equation in the form of y=mx+b. This data was stored by tapping on the File Cabinet icon.

The rate of enzyme activity for test tubes labeled 2 and 3 were analyzed by following the next steps: 10 drops of the enzyme solution were added to test tube 2, and 20 drops of solution into test tube 3. The steps used to collect data for test tube 1 were repeated for test tubes 2 and 3, separately. All three runs of data were graphed by tapping Run 3 and selecting All Runs. Each graph was displayed and this was used to collect data on Table 1a.

Experiment 2 began with an assignment of a temperature range to the group, this group was assigned 30 to 35 degrees C. All of the temperature ranges are as follows: 0 to 5 Degrees Celsius which was a 400 ml Beaker filled with ice and water, 20 to 25 degrees C, which maintained room temperature while being left alone, 30 to 35 degrees C which was put into a heated container filled with very warm water, and 50 to 55 degrees C which was a 400 ml beaker put onto a hot plate. First the three test tubes were washed, each was filled with 5 ml of 3% hydrogen peroxide and 5% water then placed into the assigned baths and left for 5 minutes. Table and Clear all Data was chosen from this menu on Lab Quest. Graph was tapped to display the graph and find the rate of enzyme activity for test tubes 1 2 and 3. One milliliter of the enzyme solution was added to test tube 2. The above steps were repeated from the previous experiment in order to record data and display a graph for oxygen gas released versus time on Lab Quest. The steps were also repeated for the remaining test tube filled with 1 mL of enzyme solution. Average rate for the three trials were recorded in Table 2a.

The third and final experiment tested the effects of pH on the rate of reaction between the enzyme and hydrogen peroxide. The test tubes were cleaned once again and placed into a test tube rack, each labeled consecutively pH 7, pH 4, and pH 10. 5 ml of 3% hydrogen peroxide and 5 ml of the corresponding pH buffer was added into each test tube. On the Lab Quest program Table was tapped and Clear all Data was chosen. After tapping Graph the Graph was displayed. 10 mL of enzyme solution Was added to each test tube and the data was collected and stored into the Lab Quest program by following the steps in experiment 1 pertaining to this action. All three runs were recorded on to a single graph after tapping Run 3 and selecting All Runs. This data was then collected and entered into Table 3a.

 

Results

The following six tables and three graphs illustrate the findings of the group’s individual data as well as class data accompanied by class average results. Each rate of reaction value is found by measuring percent of molecular oxygen released into the bottle per second as hydrogen peroxide is decomposed by the enzyme catalase. Each graph of individual findings mirrors the overall trends in class average data. This means the data is validated and conclusions can be made based on both sets of numbers.

Based on the findings, tables 1a, 1b and graph 1, the catalase is most effective at higher concentrations but does not show exponential growth, but rather a slow faster after 2mL.

Tables 2a, 2b and graph 2 show us that the temperatures between 30 and 35 degrees C cause the enzyme to bind with hydrogen peroxide most efficiently.

Finally, tables 3a, 3b and graph 3 illustrate the difference in rates of reaction between pH values 4, 7 and 10. The highest rates are seen at pH 7 and second highest at pH 10.

 

Table 1a: Effect of Catalase Concentration on Rate of Reaction with Hydrogen Peroxide

Individual Data

 

Enzyme Concentration
1 mL 2 mL 3 mL
Rate of Reaction (%/s) 0.0029865 0.022903 0.027556

 

Table 1a shows the individual data gathered by the group. When comparing the enzyme catalase concentration to rate of reaction with hydrogen peroxide it’s clear there’s a positive correlation. As the enzyme concentration goes up, so does the rate of reaction as measured by molecular oxygen being released.. There’s an interesting part of this data, between 2 mL and 3 mL the rate of reaction rises faster than between 1 mL and 2 mL.

 

Table 1b: Effect of Catalase Conc. on Rate of Reaction with Hydrogen Peroxide

Class Data

 

Enzyme Concentration        Rate of Reaction (%/s)
Group 1 mL 2 mL 3 mL
1 0.017186 0.035618 0.045212
2 0.0145 0.035 0.042
3 0 0.038957 0.057511
4 0.0029865 0.022903 0.027556
5 0.00043588 0.032031 0.046783
6 0.000077382 0.030713 0.29609
7 0.0154 0.021475 0.042819
8 0.000067473 0.032358 0.040797
Avg. Rate of Reaction (%/s) 0.006331654375 0.031131875 0.074846

 

Graph 1: Effect of Catalase Conc. on Rate of Reaction with Hydrogen Peroxide

Average Class Data

 

Table 1b/Graph 1 shows the correlation between different concentrations of the enzyme catalase and it’s reaction rate when being mixed with a solution of hydrogen peroxide. These numbers were generated by each group observing oxygen gas as hydrogen peroxide decomposes. Observe the jump between 2 mL enzyme concentration and 3 mL enzyme concentration.

Table 2a: Effect of Temps. on the Reaction between Catalase and Hydrogen Peroxide

Individual Data

 

Temperature (30-35°C)
Trial 1 Trial 2 Trial 3 Average
Rate of Reaction (%/s) 0.028994 0.028353 0.026886 0.028077

 

Table 2a illustrates the individual groups’ data in the temperature experiment. After three trials of measuring the rate of reaction between the enzyme catalase and hydrogen peroxide at 30 to 35 degrees C, an average rate of reaction was found to be at 0.028077 %/s.

 

Table 2b: Effect of Temps. on the Reaction between Catalase and Hydrogen Peroxide

Class Data

 

Temperature (°C)   &   Rate of Reaction (%/s)
0-5 20-25 30-35 50-55
0.0055564 0.037567 0.05908 0.040668
0.014701 0.035509 0.063534 0.040803
0.0079987 0.033145 0.047107 0.046642
0.018266 0.032135 0.028994 0.0357
0.014822 0.039001 0.028353 0.03032
0.080515 0.02389 0.026886 0.0335
Average Rate of Reaction (%/s) 0.02364318333 0.03354116667 0.04232566667 0.03793883333

 

Graph 2: Effect of Temps. on the Reaction between Catalase and Hydrogen Peroxide

Average Class Data

 

Table 2b/Graph 2 shows us each groups three trials (two groups to each temp. range). A peak rate of reaction is found around 30 to 35 degrees C. From 0 to 25 degrees C, the rate is slowly rising. Ater 30-35 degrees C, the rate of reaction falls.

 

Table 3a: Effect of pH values on the Reaction between Catalase and Hydrogen Peroxide

Individual Data

 

pH
4 7 10
Rate of Reaction (%/s) 0.023885 0.0259454 0.025282

 

Table 3a shows the correlation between pH values as defined by a buffer added to the solution and the rate of reaction as catalase reacts with hydrogen peroxide. The enzyme is most effective between pH values of 7 and ten. The highest rate of reaction was observed at pH of 7.

 

Table 3b: Effect of pH values on the Reaction between Catalase and Hydrogen Peroxide

Class Data

 

pH values    &    Rate of Reaction (%/s)
Group 4 7 10
1 0.01473 0.032514 0.034101
2 0.014061 0.03235 0.025647
3 0.02608 0.031551 0.019802
4 0.032273 0.034514 0.034509
5 0.023885 0.025945 0.025283
6 0.027562 0.03492 0.032833
7 0.0345 0.0365 0.034
8 0.025316 0.038689 0.03829
Average Rate of Reaction (%/s) 0.024800875 0.033372875 0.030558125

 

Graph 3: Effect of pH values on the Reaction between Catalase and Hydrogen Peroxide

Average Class Data

 

Table 3b/Graph 3 shows rate of reaction measured for each pH value. Although the numbers vary from the individual data gathered, the trend is the same. The highest rate of reaction is observed at a pH of 7 while the second highest value is at pH of 10. The lowest rate of reaction is found at pH 4.

 

Discussion

The hypothesis for different concentrations of the enzyme catalase was supported when predicting the oxygen being decomposed from hydrogen peroxide. It was predicted that the amount of oxygen sensed would go up based on the amount of catalase present I also predict that the rate of reaction would reach saturation. This means that the catalase concentration yielded proportionate amount of molecular oxygen based on the amount of catalase. The graph of this data plateaus. Physically, the enzymes have binding sites where components of hydrogen peroxide can bind and then be broken  down by the protein. The more enzyme present means more binding sites ready to react with the chemical. Our bodies must need a set amount of catalase based on hydrogen peroxide it expects to encounter in the body and not a substantial amount more because more of the enzyme does not mean a higher rate of reaction. (2)

The hypothesis for the effect of temperature on oxygen being sensed was also accepted as hydrogen peroxide breaks down because of the catalase. It was hypothesized that the highest rate of reaction would be found at 30 degrees to 35 degrees. This was true because the temperature of mammalian bodies is in this range of temperatures.(2) Catalase, and in this case cow liver catalase, shows to be the most effective in an environment closest to the one it was created to function in.

The hypothesis for the amount of oxygen released based on ph was confirmed. The highest rate of oxygen broken from the hydrogen peroxide occurred at the pH 7. This is the pH closest to body temperature, where the enzyme catalase would be used two functioning in and created to function in the body. The pH of for yielded lower results and so did the pH of 10. Although it was observed that the pH of 10 gave a higher rate of reaction value than the pH of 4. This leads to the belief that our bodies maintain a pH close to 7 in many compartments of the body in order to function properly. In this case the body needs catalase to break down the toxic hydrogen peroxide and make oxygen and water which are non toxic to our bodies.

Although our bodies use this enzyme to decompose the chemical into molecules that are not harmful to us in these conditions, it is important to note that too much oxygen created by this process is harmful. Oxygen levels that increase due to the reaction of catalase and hydrogen peroxide can build up in compartments in our bodies such as in the brain and veins. In the case that a solution containing more than 3% hydrogen peroxide is ingested, embolisms or oxygen ‘bubbles’ can be caused in these tissues. (3) This is an example of the enzyme functioning efficiently, but overproduction of the by product causing health issues.

 

References

  1. Evans CA. On the Catalytic Decomposition of Hydrogen Peroxide by the Catalase of Blood. Biochem J. 1907;2(4):133–155.

 

  1. Williams J. THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE. The Journal of General Physiology. 1928;11(4):309-337.

 

  1. Sung J, Cossarini F, Palaiodimos L, Benson B, Meholli M. Extra Oxygen Leads to Bubble Trouble: Portal Vein Gas Embolism from 3% Hydrogen Peroxide Ingestion. Muacevic A, Adler JR, eds. Cureus. 2018;10(2):e2136. doi:10.7759/cureus.2136.

 

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