Amelia Houlihan - Catalase Lab Report PDF

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This is one of two mandatory lab reports for the biology 101 lab class. This section was taught by Professor Levina....

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The Effects of Catalase Temperature and Substrate Concentration on O2 Reaction Rates Amelia Houlihan Section 10 September 26th, 2017 Devin Carr

Abstract An enzyme is a protein that catalyzes chemical reactions by binding to a substrate. This experiment utilizes catalase as the enzyme and uses hydrogen peroxide as the substrate that it breaks down. The experiment had two parts to it. In the first part, the hypothesis was that as substrate concentration increased, so would the reaction rate until all active sites are bound. In the second part, it was hypothesized that as temperature increased, so would the reaction rate until the enzyme is denatured. Catalase was mixed with various substrate concentrations in the first part, then mixed catalase at various temperatures with a 0.8% substrate concentration in the second half of the experiment to measure the O2 output. The varied substrate concentrations of 0.0%, 0.1%, 0.2%, 0.4%, and 0.8% produced averaged reaction rates of 0, 0.73, 2.18, 18.74, and 44.55 ml/min respectively. The varied temperatures of catalase, from coolest to warmest, produced averaged reaction rates of 79.98, 48.55, 93.27, and 3.80 ml/min. The results supported both hypotheses proving that increasing substrate concentration and temperature will also increase the reaction rate. In both cases, when the active sites are filled or the temperature is increased too greatly, the enzyme will become inactive. Introduction Enzymes are protein catalysts whose function is to speed up a chemical reaction without any amount of the enzyme being used up. Although metabolic pathways are capable of selfregulation, enzymes decrease the amount of activation energy needed to start a reaction, therefore increasing the reaction rate (Hammes & Wu, 1971). This experiment utilized the enzyme Catalase. Catalase’s specific function is to break down hydrogen peroxide (H2O2) into H2O and O2 and was first discovered to be present in living cells when it was taken from the leaf of a tobacco plant (Ranjan & Malik, 1931). Many initial experiments attempted to find a correlation between enzyme activity and respiration. However, it was concluded that the usage of catalase is to prevent excessive oxidation, not to assist in respiratory metabolism (Ranjan & Malik, 1931). The purpose of this experiment was to observe how varying the temperature of the catalase and varying the substrate concentration affected the reaction rate of O2. The null hypotheses were that as the substrate concentration increased, so would the reaction rate until all the active sites were bound, and that as the temperature of the catalase increased, so would the

reaction rate until the enzyme was denatured. The alternative hypotheses were that the varied substrate concentrations and varied temperature would have no effect on reaction rates. The catalase speeds up the reaction proportional to the amount of substrate added. Once the active sites of the catalase are filled, it will no longer function to catalyze the reaction. Likewise, enzymes work at very specific temperatures, so they will catalyze a reaction up to a certain temperature, and then they will begin to denature and become inactive (Aronson, Fraser, Smith, 1956). Materials and Methods For each test that was conducted, a 600 ml beaker was filled with water. A 10 ml graduated cylinder was filled with water, sealed with a thumb, inverted, and immersed in the beaker. A U-shaped glass tube was inserted into the opening of the graduated cylinder. This experiment began by creating a control group that did not use catalase. A phosphate buffer of 10 ml was added to 10 ml of a 0.8% concentration of H2O2 in a 50 ml Erlenmeyer flask. The rubber stopper on the opposite end of the tube was put in place to seal the solution in the flask. The flask was swirled until 10 ml of O2 was produced and the reaction rate produced per minute was recorded. This procedure was repeated, in the first part of the experiment, with varying concentrations of substrate. These concentrations were 0.8%, 0.4%, 0.2%, 0.1%, and 0.0%. Each concentration was combined with regular, room temperature, catalase and swirled to produce 10 ml of O2. In the second half of the experiment, the same procedure was used but the substrate concentration remained the same at 0.8%, while the Catalase temperature was varied. Boiled catalase, catalase in a warm water bath (40oC), room temperature catalase (25oC), and catalase on ice (0oC) were the temperatures used. Each was combined with the 0.8% substrate and swirled to produce 10 ml of O2. All data regarding reaction rates in ml per second were recorded. Microsoft

Excel was used to create graphs and tables. The p-value cutoff was 0.05. A t-test was used to discover any statistical significance.

Results The substrate concentration of 0.0% did not elicit a reaction, therefore no O2 was produced. The substrate concentration of 0.1% produced an average of 0.73 ml of O2 per minute. The concentration of 0.2% produced an average of 2.18 ml of O2 per minute, and the concentrations of 0.4% and 0.8% produced an average of 18.74 and 44.55 ml/min respectively (Table 1). The average reaction rate for the catalase on ice was 79.98 ml/min. When the catalase temperature was raised to room temperature, an average of 48.55 ml of O2 per minute was produced. When the catalase was warm it produced an average of 93.27 ml of O2 per minute, and the boiled catalase produced an average of 3.80 ml of O2 per minute (Table 2). A t-test was conducted to determine the statistical significance between each temperature condition and room temperature. The p-value for room temperature versus warm temperature was 0.002. The p-value for room temperature versus cold (0oC) was 0.014. The p-value for room temperature versus boiled was 1.47E-07 (Table 3).

Table 1: Effect of change in substrate concentration on reaction rate Substrate concentration

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group 7

Group 8

Average

Std dev

0.0%

0

0

0

0

0

0

0

0

0.00

0

0.1%

1.5

1

0.4

0.55

0.75

0.8

0.84

0

0.73

0.411916254

0.2%

3.05

3.5

1.2

2

3

2.2

2.5

0

2.18

1.061819635

0.4%

10

20

18.18

21.73

35

20.53

12.77

11.69

18.74

7.417580047

21.7

60

43.48

23.25

60

46.3

50

51.68

44.55

13.86526364

0.8%

Table 1- Statistics table for comparing average reaction rate times based on varied substrate concentrations.

Table 2: Effect of change in temperature on reaction rate in a catalase mediated breakdown of H2O2 Temperature factor

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group 7

Group 8

Average

Stdev

Cold (0oC) Room Temperature(25oC)

100

120

45.87

90.9

100

89.76

48.08

45.25

79.98

27.40

58

42.86

43.48

23.25

60

59.13

50

51.68

48.55

11.41

Warm (37oC)

90

150

68.03

100

120

100

62.5

55.66

93.27

29.65

10

12

0

3.84

0

0

4.59

0

3.80

4.53

o

Boiled (100 C)

Table 2- Statistics table for comparing average reaction rate times based on varied catalase temperatures. Table 3: Analysis of statistical significance for each temperature condition vs room temperature (t-test: two sample assuming equal variances) temperature comparison used for p values t-test statistical analysis p value for room temp vs warm temperature p value room temp vs cold p value room temp vs boiled

0.002264892 0.014128458 1.46767E-07

Table 3- T-test and p-values to demonstrate statistical significance for temperature conditions

Reaction Rate ml/min

Effect of Substrate Concentration on Reaction Rate 50 45 40 35 30 25 20 15 10 5 0 0.0%

0.1%

0.2%

0.3%

0.4%

0.5%

0.6%

0.7%

0.8%

0.9%

Substrate Concentration

Figure 1 – Reaction rate of O2 based on varied percentages of substrate concentration. Error bars show one standard deviation.

Reaction rate ml/min

Effect of Temperature on Reaction Rate 100.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00

Cold (0oC)

Room temp (25oC)

Warm (40oC)

Boiled (100oC)

Catalase Temperature

Figure 2- Reaction rate of O2 based on varied temperatures of catalase. Error bars show one standard deviation.

Discussion The purpose of this experiment was to test the effects of varied substrate concentrations and varied catalase temperatures on the reaction rate of O2. It tested the hypothesis that as substrate concentration increased, so would the reaction rate until all the active sites were bound. It also aimed to test the hypothesis that as temperature increased, so would the reaction rate until the enzyme was denatured. The reaction rate increased from 0 ml/min to 44.55 ml/min as the substrate concentration was increased from 0.0% to 0.8%. This statistic supports the hypothesis for part one of the experiment because the substrate concentration did cause the reaction rate to increase accordingly. As the substrate concentration increases, more substrate molecules are combining with more enzyme molecules, thus creating a greater amount of product and increasing the rate of the reaction. In the second part of the experiment, the data showed an increase of reaction rate as the temperature increased, but then dropped when the temperature was increased to 100oC.

According to the hypothesis, the reaction rate should have sped up as the temperature increased, until the enzyme denatured, which is exactly what happened. The enzyme was heated too much, causing it to become inactive, and no longer effect the speed of the reaction (Aronson, Fraser, Smith, 1956). The p-value for room temperature versus warm temperature catalase was 0.002. The p-value cut-off was 0.05, which means that there was a significant difference between the reaction rates of the varied catalase temperatures. Similarly, the p-value for room temperature catalase versus cold catalase was 0.014. Since this number is also less than the p-value cut-off of 0.05, there is a statistical significance between the reaction rates. As it relates to the boiled catalase, there was a difference in the reaction rates, however, for this temperature the reaction rate decreased instead of increasing because the enzyme was denatured. Limitations that may have affected the results of the experiment are inability to control other student’s data, inaccurate measurements of the catalase or the substrate, and inconsistent swirling of the solution, all due to human error. For future experiments, making sure each group’s measurements are the same and having one person swirl all the solutions may produce more accurate data. Works Cited Aronson, D.L., Fraser, M.J., and Smith, C.L. (1956) Enzyme Alteration by Ionizing Radiation. Radiation Research 5(3): 225-237 Hammes, G. and Wu, C. (1971) Regulation of Enzyme Activity. Science 172(3989): 1205-1207 Ranjan, S. and Malik, A.K. (1931). A Study of the Catalase Reaction, with Special Reference to Respiration in Plants. The New Phytologist 30(5): 355-357....