Catalytic activity of the enzyme catalase demonstrates high efficiency at neutral temperatures and a neutral p H level PDF

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Catalytic activity of the enzyme catalase demonstrates high efficiency at neutral temperatures Lab and a neutral p H level Lab Bio 1...

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Biology Lab Sec 07 24 October 2017 Catalytic activity of the enzyme catalase demonstrates high efficiency at neutral temperatures and a neutral pH level Introduction Enzymes are biological molecules which accelerate chemical reactions without being consumed by the reaction. Typically, enzymes are proteins that function to decrease activation energy in chemical reactions (5). Hundreds of different enzymes are produced by cells and are created in a customize way to perform the necessary reactions for life. Each enzyme is individualized to recognize one substrate, which is the molecule the enzyme binds to, to generate a specialized product. Within the enzyme, there are active sites which lie within the enzyme folds. The active site serves as a meeting place for the substrate and enzyme to come together in order to form a chemical reaction. The efficiency of the enzyme is altered by conditions such as temperature and pH level (1). In this experiment, the enzyme “catalase” was observed. The role of catalase is to breakdown hydrogen peroxide as well as protect the cells from oxidative damage. The structure of catalase is uniquely derived from the demand to rid hydrogen peroxide from aerobic organisms. Hydrogen peroxide is a unique toxin because it is stable in abiotic environments at ambient temperature and neutral pH, but rapidly kills any kind of cell (2). A catalase reaction with hydrogen peroxide primarily involves the absorption of hydrogen peroxide at the catalase surface. The decomposition of hydrogen peroxide by catalase is a maximum at the optimum pH 6.8 to 7.0 (3). During experimentation, the decomposition of hydrogen peroxide and efficiency of catalase were observed when 60% potato extract was combined with 3% hydrogen peroxide and tested at various temperatures as well as pH (1). When hypothesized, the enzyme, catalase, will be most efficient at an ambient temperature of 25°C and a neutral pH. Materials and Methods In part 1 of the catalytic enzyme activity, the concentration of 60% was determined to be the concentration of focus. This concentration was prepared by creating the appropriate dilution. Using a graduated cylinder, 8 mL of buffer was measured and transferred into a beaker. Following, 12 mL of potato extract was added to the same beaker as the buffer. The solutions were then mixed with a transfer pipette. Next, 2 mL of 3% hydrogen peroxide was added to the test tube labeled for the 60% concentration. After this task was performed, 2mL of the 60% dilution was transferred into the test tube containing the hydrogen peroxide. The reaction then took place and sat at room temperature for a total of 5 minutes. The volume was then measured from the top of the liquid to the top of the foam. This is how the 60% concentration was created and determined. In part 2 of the catalytic enzyme activity, the variables of temperature and pH were tested with catalase. Experimentation began with testing temperature. Four test tubes labeled 0°C, 25°C, 37°C, and 95°C were obtained and 2mL of 60% enzyme was added to each test tube. The test tubes containing the enzyme were then placed in the appropriate temperature and incubated for a total of 5 minutes. After the 5 minutes, 2 mL of 3% hydrogen peroxide was added to each test tube containing the enzyme. Again, incubation of each test tube at the correct temperature

took place for a total of 5 minutes. The volume was then taken from the top of the liquid to the top of the foam and recorded in the designated table. Following temperature, the pH procedure was then performed. Three test tubes labeled pH 3, pH 7, pH 11 were obtained. Then, 3 mL of 60% catalase enzyme was added to each of the test tubes. Next, 3 mL of the appropriate pH buffer was added to the corresponding test tube. The test tubes were then incubated at room temperature (25°C) for 5 minutes. The foam was then measured from the top of the liquid to the top of the foam and recorded in the designated table. Results When analyzing the effects of temperature on catalase activity based off the class average, the data revealed that the highest temperature (95°C) produced the least amount of foam. This led to a conclusion that the enzyme is least active at high and extreme temperatures. The greatest amount of foam was produced at a temperature of 25°C followed by 37°C and then 0°C. Figure 1 displays the catalase activity as increasing from temperature 0°C -25°C, peaking at 25°C, and then decreasing from 25°C -95°C. The range (based off the class average) was 5.231 mL. The results showed that 25°C produced the greatest foam value of 5.263 (Table 1); therefore, the enzyme is most active at a neutral (or room) temperature as shown in Figure 1. Table 1: Effect of temperature on catalase activity Foam Volume Incubation Temperature (°C) (mL) by group 0 25 37 Bench group 4 5.6 5.75 Class average 3.781 5.263 3.975

95 0 0.032

Figure 1: Effects of Temperature on Catalase Activity

Effect of Temperature on Catalase Activity 6 5

Volume (mL)

4 3 2 1 0 0

10

20

30

40

50

60

Temperature (°C ) Class Average Volume (mL)

70

80

90

100

Figure 1: Graph displaying the class average volume for the effects of temperature on catalase activity at 60% concentration When analyzing the effects of pH on catalase activity based off the class average, the results revealed that a pH of 3, a more acidic pH, produced the least or no foam volume. In contrast, a neutral pH of 7 produced the greatest amount of foam with an average of 3.026 (Table 2). When analyzing Figure 2, the catalase activity is shown to be increasing between the pH of 37, reaching a peak at a pH of 7, and decreasing from the pH of 7-11. According to Figure 2, catalase activity is most active at a neutral pH of 7. Table 2: Effect of pH on catalase activity Foam Volume (mL) 3 by group Bench group 0 Class average 0

pH 7 2.5 3.026

11 1.75 1.596

Figure 2: Effects of pH on Catalase Activity

Effect of pH on Catalase Activity 3.5 3

Volume (mL)

2.5 2 1.5 1 0.5 0

2

3

4

5

6

7

8

9

10

11

12

pH Class Average Volume (mL)

Figure 2: Graph displaying the class average volume for the effects of pH on catalase activity at 60% concentration Discussion/Conclusion When analyzing the data from Figures 1 and 2, as well as Tables 1 and 2, the hypothesis of enzyme activity to be greatest at 25°C as well as a neutral pH was supported. The effects of temperature on catalase activity (Figure 1; Table 1), activity is shown to be paramount at a temperature of 25°C with an average of 5.263 mL of foam volume. Furthermore, the temperature of 25°C is an ambient temperature meaning it is relating to the temperature of the surroundings, in this case room temperature. Additionally, the effects of pH on catalase activity (Figure 2;

Table 2), displayed catalase activity to be maximum at a pH of 7 which is neutral. In all, experimental data supports the subject hypothesized. From previous information, the role of catalase is to breakdown hydrogen peroxide. As stated, the decomposition of hydrogen peroxide by catalase is a maximum at the optimum pH 6.8 to 7.0 (3). This was observed through tests of pH on enzyme activity and verifies prior knowledge of the catalase interaction with hydrogen peroxide. When catalysis occurred, oxygen was produced, and bubbles of oxygen gas were created in a foam-bubble form in the test tube (1). This was how the reaction and efficiency of the catalase was measured for both pH and temperature. Therefore, the temperature and pH with the greatest foam volume yielded to the greatest environment for catalase activity. Limitations on experimentation could have been possible. Primarily, this laboratory tested for enzyme activity in potato extract, therefore, it cannot determine a definite optimal pH and temperature for all catalase enzyme activity. Optimal enzyme activity may vary by substance for the conditions of temperature and pH. For example, when investigating enzyme function in relation to hypothermia and platelet regression, temperatures below 33°C resulted in reduced platelet function as well as reduced enzyme activity (4). An additional limitation could have included the limited variables tested for the temperatures and pH. Four temperatures were tested for the optimal temperature, but the temperatures were widespread, therefore the activity could have been greater at a temperature note tested. Furthermore, the same observation applies to pH due to only three pH’s being tested. This experiment holds importance because of its relation to medicine as well as scientific knowledge. Through the analyzation of enzymes, medical discoveries are able to be made. For example, a study has hypothesized hypothermia-associated bleeding in relation to core temperature has resulted from dysregulation of enzymatic function (4). Therefore, by understanding the relation between core temperature and enzymatic function, many medical problems such as coagulopathy have been able to create medical advancements. References 1. Duquesne University. “Catalytic activity of Enzymes Part 2,” Biology 111L Lab Manual. 2017. 2. Mahaseth, T., Kuzminov, A. 2016. Potentiation of hydrogen peroxide toxicity: From catalase inhibition to stable DNA-iron complexes. PubMed. 773(10):274-281. 3. Williams J. 1928. THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE. The Journal of General Physiology.;11(4):309-337. 4. Wolberg, A., Meng, Z., Monroe, D., Hoffman, M. 2004. A Systematic Evaluation of the Effect of Temperature on Coagulation Enzyme Activity and Platelet Function. The Journal of Trauma and Acute Care Surgery. 56(6):1221-1228. 5. Eed, J. 2013. Factors Affecting Enzyme Activity. ESSAI: 10(19):1....