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The Influence of Temperature and PH on the Activity of an Enzyme

Amy Gustafson Aaron Rick BI 102: Cell Biology and Genetics Section: 001 Dr. Batiza October 18, 2011

Abstract: This experiment tested the activity of a substrate reaction when using an enzyme. The effect of changes in temperature and changes of PH on the activity of an enzyme were both analyzed. The enzyme used was crude potato extract and the substrate used was hydrogen peroxide (H2O2). To test the change in temperatures, one reaction had both the enzyme and the substrate in a hot bath, in an ice bath, and in room temperature. To test the change in PH, phosphate buffers with high, low and neutral PH were added to the reaction. The results of this experiment did not show a significant difference in the enzyme activity with changing temperatures. The enzyme activity with changes in PH were significant with a decrease in PH but were not significant with an increase in PH. This does not match the expected results of the test and further experimentation needs to be done because the results were fairly inconclusive.

Introduction: To speed up the process of some reactions, an enzyme is used. An enzyme is a protein that speeds up the reaction by lowering the activation energy required to start the reaction (1). Different factors can affect how the enzyme is working because enzymes have an optimum temperature range as well as an optimum PH range (1). If the temperature exceeds the optimum temperature, the enzyme will begin to denature, or come apart, decreasing the activity of the enzyme (1). If the temperature is too low, the substrates, the molecules the enzyme acts upon, will be moving slowly (1). This decreases the likelihood of a collision between the enzyme and the substrate thus decreasing the activity of the enzyme (1). With a change in PH, the enzyme itself is altered (1). The shape of the protein can change due to the different amino acids that

create the protein (1). With a new shape, the substrate may no longer be able to bind to the enzyme (1). This would decrease the activity of the enzyme as well (1).

For this lab, the substrate used was hydrogen peroxide (H2O2). The enzyme used was crude potato extract. The purpose of this lab was too become familiar with different ways enzyme activity can be altered by establishing two hypotheses. The hypothesis tested were as follows: A change in temperature will cause a change in the reaction rate of hydrogen peroxide (H2O2) when using crude potato extract as an enzyme and a change in PH will cause a change in the reaction rate of hydrogen peroxide (H2O2) when using crude potato extract as an enzyme.

Methods: To test the affect that temperature had on the enzyme, the potato was skinned and cut into pieces. It was then blended until it became pureed. The liquid was then drained out of the pureed potato by placing a cheese cloth over a beaker and taping the cheese cloth to the beaker to ensure it did not fall in. Once it was completely drained, the crude potato extract was ready to be used as an enzyme. Half a milliliter of the enzyme was placed into three different test tubes. Then one milliliter of the substrate, hydrogen peroxide (H2O2), was measured into three different test tubes as well. One test tube with enzyme and one test tube with hydrogen peroxide (H2O2) were placed in a hot bath with a temperature of 98C. Then one test tube with enzyme and one test tube with hydrogen peroxide (H2O2) were placed in an ice bath with a temperature of 7C. The remaining two test tubes, one with the enzyme, crude potato extract, and one with the substrate, hydrogen peroxide (H2O2), were left to sit in room temperature, about 25C. After five minutes, the test tubes of the substrate, hydrogen peroxide (H2O2), were dumped into the test tubes of the enzyme

with the same temperature. The height of the foam from the reaction was then measure, in centimeters, using a ruler, at 30, 60 and 90 seconds. Three trials of the warm temperature, cold temperature and room temperature reactions were conducted.

To test the affect that PH had on the enzyme, the potato was skinned and cut into pieces. It was then blended until it became pureed. The liquid was then drained out of the pureed potato by placing a cheese cloth over a beaker and taping the cheese cloth to the beaker to ensure it did not fall in. Once it was completely drained, the crude potato extract was ready to be used as an enzyme. Half a milliliter of the enzyme was then placed into three separate test tubes. One milliliter of the high PH (9) additive was placed in one test tube, one milliliter of the low PH (4) additive was placed into one test tube, and one milliliter of the neutral PH (7) additive into the final test tube. The additives used were phosphate buffers. The enzyme and the additive were then mixed together by gently swirling the test tubes. Then, one milliliter of the substrate, hydrogen peroxide (H2O2), was added to the mixture. The height of the foam from the reaction was then measure, in centimeters, using a ruler, at 30, 60 and 90 seconds. Three trials of the high PH, low PH, and neutral PH reactions were conducted.

Results: To determine if a change in temperature or a change in PH would affect the enzyme, three measurements were taken for each trial. For the t-test conducted to compare the activity of the enzyme, only the 90 second measurement was used due to the fact that this was the end of the reaction.

For the temperature reaction, a high temperature at 98C, a room temperature at 25C, and a low temperature at 7C were used to compare how temperature affects the activity of an enzyme (Table 1). The average height of the high temperature reaction was 2.2 centimeters, the average height of the room temperature reaction was 5.97 centimeters and the average height for the low temperature reaction was 6.57 centimeters (Figure 1). The standard deviation of the high temperature reaction was 0.1 centimeters, the standard deviation of the room temperature reaction was 4.76 centimeters and the standard deviation of the low temperature reaction was 2.1 centimeters (Figure 1). When comparing the high temperature results to the room temperature results, a p-value of 0.242 was obtained. When comparing the low temperature results to the room temperature results, a p-value of 0.851 was obtained.

Table 1: This is the table of all the data collected for the various temperatures throughout the course of the experiment. For the analysis, only the 90 second interval was used because this measurement was taken after the enzyme had finished reacting.
High Temperature (98C) Trial 30 sec 60 sec 90 sec 1 2 2.1 2.1 2 2.4 2.3 2.2 3 2.3 2.3 2.3 average: 2.233333 2.233333 2.2 standard deviation: 0.1 Room Temperature (25C) 30 sec 60 sec 90 sec 3 4.4 5.1 1.7 1.7 1.7 8.2 10.6 11.1 4.3 5.566667 5.966667 4.759552 Low Temperature (7C) 30 sec 60 sec 90 sec 4 6.6 8.6 2.5 3.2 4.4 2.8 4.9 6.7 3.1 4.9 6.566667 2.103172

Temperature
Height of the foam of the reaction (cm)

12

10 8 6 4 2 0 High Temperature (98) Room Temperature (25) Low Temperature (7)

Temperature of Enzyme and Substrate (C)

Figure 1: This is the graphical representation of the averages of the 90 second measurements at the high, low, and room temperature measurements. The standard deviations of these three measurements are also displayed with the error bars.

For the PH reaction, a high PH of 9, a neutral PH of 7, and a low PH of 4 were used to compare how PH affects the activity of an enzyme (Table 2). The average height of the high PH reaction was 7.3 centimeters, the average height of the neutral PH reaction was 6.27 centimeters and the average height for the low PH reaction was 3.13 centimeters (Figure 2). The standard deviation of the high PH reaction was 0.173 centimeters, the standard deviation of the neutral PH reaction was 0.666 centimeters and the standard deviation of the low PH reaction was 0.208 centimeters (Figure 2). When comparing the high PH results to the neutral PH results, a p-value of 0.06 was obtained. When comparing the low PH results to the neutral PH results, a p-value of 0.001 was obtained.

Table 2: This is the table of all the data collected for the various PH values throughout the course of the experiment. For the analysis, only the 90 second interval was used because this measurement was taken after the enzyme had finished reacting.
High PH (9) Neutral PH (7) Low PH (4) 30 sec 60 sec 90 sec 30 sec 60 sec 90 sec 30 sec 60 sec 90 sec 4.8 6.7 7.4 3.5 4.5 5.5 2.8 2.9 2.9 2 4.3 6.1 7.4 4.7 5.3 6.7 3.1 3.3 3.3 3 4.9 6 7.1 3.9 5.4 6.6 2.9 3 3.2 average: 4.666667 6.266667 7.3 4.033333 5.066667 6.266667 2.933333 3.066667 3.133333 standard deviation: 0.173205 0.665833 0.208167 Trial

PH Values
Height of foam of reaction (cm)
8 7 6 5 4 3 2 1 0

High PH (9)

Neutral PH (7)
PH of Additive

Low PH (4)

Figure 2: This is the graphical representation of the averages of the 90 second measurements at the high, low, and neutral PH measurements. The standard deviations of these three measurements are also displayed with the error bars.

Discussion: One hypothesis tested was a change in temperature will cause a change in the reaction rate of hydrogen peroxide (H2O2) when using crude potato extract as an enzyme. With just looking at the averages of the 90 second results, no conclusions can be drawn from that information. When

looking at the standard deviation for the 90 seconds results, it is evident that there was a great amount of variability with the room temperature and low temperature results. The p-values must be analyzed to determine if the results obtained were significant. When comparing the high temperature results to the room temperature results, a p-value of 0.242 was obtained. When comparing the low temperature results to the room temperature results, a p-value of 0.851 was obtained. With p-values greater than 0.05, the hypothesis that a change in temperature will cause a change in enzyme activity is rejected. This experiment provided no evidence for a difference in enzyme activity with different temperatures. However, there were some confounding variables that played a role in the results. While the tests of temperature were conducted, the enzyme was simply sitting out in room temperature. This could have caused some natural decrease in activity of the enzyme itself. There is also the human error in measurements of the enzyme, crude potato extract, and the substrate, hydrogen peroxide (H2O2), which would have led to various beginning heights as well as differences in the reaction due to varying concentrations of each component. Human error in the measuring of the height of the foam also must be taken into account. Another error may have been present in the room temperature tests. The results are all over the board and this group had a relatively high standard deviation when compared to other results. The reason behind this is unknown, but may have affected the results of the experiment as a whole.

The other hypothesis tested was a change in PH will cause a change in the reaction rate of hydrogen peroxide (H2O2) when using crude potato extract as an enzyme. With just looking at the averages of the 90 second results, no conclusions can be drawn from that information. When looking at the standard deviation for the 90 seconds results, it is evident that there was the greatest amount of variability with the neutral PH results. The p-values must be analyzed to

determine if the results obtained were significant. When comparing the high PH results to the neutral PH results, a p-value of 0.06 was obtained. When comparing the low PH results to the neutral PH results, a p-value of 0.001 was obtained. With one p-value greater than 0.05 and one p-value lower than 0.05, the hypothesis that a change in PH will cause a change in enzyme activity is rejected. However, a p-value of 0.06 is very close to being a significant difference, so additional tests should be run to investigate if it is indeed significant. This experiment provided no evidence for a difference in enzyme activity with a higher PH, but provided evidence for a difference in enzyme activity with a lower PH. However, there were some confounding variables that played a role in the results, just as in the temperature experiment. While the tests of temperature were conducted, the enzyme was simply sitting out in room temperature. This could have caused some natural decrease in activity of the enzyme itself, especially because all the PH tests were taken after the temperature tests were completed. There is also the human error in measurements of the enzyme, crude potato extract, and the substrate, hydrogen peroxide (H2O2), as well as the additive that was used to change the PH of the reaction. All of these would have led to various beginning heights as well as differences in the reaction due to varying concentrations of each component. Human error in the measuring of the height of the foam also must be taken into account.

Overall, the test of change of temperature turned out to be rejected while the test of change of PH was somewhat inconclusive because one value was significant while the other was not. Neither resulted was what was expected with the research that changes of temperature and changes of PH would affect the activity of the enzyme (1). Further experiments should be conducted to minimize the errors and improve the methods that were used to extract the data. With further

experiments and more trials to base the conclusions on, a significant difference may be discovered.

References: 1. Raven, Johnson, Mason, Losos, and Singer. Foundations of Life- Chemistry, Cells and Genetics. 9th ed. The McGraw-Hill Companies, Inc., 2011. Print.

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