Procedure 2: Effect of pH
Research Question: In this lab my partner and I were determining what effect pH had on enzymatic activity. We had many pH amounts to choose from and we were determined to find which pH amount could produce the fastest reaction.
Hypothesis: After observing the pHs available we came to a hypothesis that a median pH can result in the most enzymatic activity. pH values that are too high or too low can have a slow effect on enzymatic activity. With this hypothesis in mind we began working on our experiment.
Procedure: In this lab we had five pairs of tubes totaling of 10 tubes. With each pair we labelled one tube enzyme and another tube substrate. To the substrate tubes we added 7 ml of distilled water, 0.3 mL of 0.1 percent hydrogen peroxide, and 0.2 mL of guaiacol for a total volume of 7.5 mL. To the enzyme tubes we changed the amount of pH added. We added 6.0 ml of a specific pH and 1.5 mL of peroxidase for a total volume of 7.5 mL. We used pHs 3, 5, 7, 8, and 10. After making the enzyme and substrate solutions we covered the tubes with parafilm until we were ready to combine them. Once we were ready we took one enzyme solution and one substrate solution and combined them into one tube labeled reaction. We covered the tube with the parafilm once again, inverted it twice to mix, and placed it on the tube rack. We timed the reaction for three minutes and recorded observations and took pictures of the solution’s color every 30 seconds. After the trial was over we compared our pictures and observations to the color chart.
Data:
Above is a graph showing the Progression of color values of each pH we used over the three minute trial. The x axis represents time and the y axis represents the color chart. Each line represent a different pH we used. For example, the blue line shows the progression of color of the enzyme with pH 7.
Claim: Having an intermediate pH can produce the most enzymatic activity compared to a pH that is too high or too low which would produce slow enzymatic activity.
Evidence:
Color Chart
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Tube
|
pH
|
0(sec)
|
30(sec)
|
60(sec)
|
90(sec)
|
120(sec)
|
150(sec)
|
180(sec)
|
1
|
7
|
2
|
3
|
5
|
6
|
8
|
9
|
10
|
2
|
5
|
1
|
4
|
6
|
8
|
9
|
10
|
10
|
3
|
8
|
1
|
4
|
5
|
6
|
7
|
8
|
10
|
4
|
3
|
1
|
1
|
1
|
1
|
2
|
2
|
2
|
5
|
10
|
1
|
1
|
1
|
1
|
1
|
2
|
2
|
Above is shown our data chart. Each tube we used is shown alongside its pH value. Over the 30 second increments the value reached from the color chart is shown.
Reasoning: Tube 2 which had a pH of 5 had the fastest reaction. By 150 seconds it reached 10 on the color chart. Other tubes that had relatively intermediate pH’s like tube 1 with pH 7 and tube 3 with pH 8 also had fast enzymatic activity. By the end of the 3 minute trial all of these tubes had reached 10 on the color chart. Tube 4 had a low pH of 3 and by the end of the 3 minute trial it reached 2 on the color scale. Too low of a pH does not allow the process to function its best. Tube 5 with a high pH of 10 also had slow enzymatic activity and reached 2 by the end of the 3 minute trial. Too high of a pH can denature the enzyme therefore slowing the reaction.
Procedure 3: Independent Investigation
Research Question: My partner and I now had some experience with the enzyme and substrate reactions. We decided to investigate the substrate solutions a little more. We decided to research how does changing the substrate concentration affect enzymatic activity.
Hypothesis: We hypothesized that increasing the substrate concentration would increase the enzymatic activity during a reaction. With this in mind we began our experiment.
Procedure: We took 4 pairs of tubes. Within each pair we labelled one enzyme and one substrate. The enzyme solutions all had 6.0 mL of distilled water and 1.5 mL of peroxidase for a total volume of 7.5 mL. Each tube that was labelled substrate were all different from one another because each had different substrate concentrations. Tube 1 had 1 mL of hydrogen peroxide, 0.2 mL of guaiacol, and 6.3 mL of water. Tube 2 had 0.2 mL of hydrogen peroxide, 0.2 mL of guaiacol, and 7.1 mL of water. Tube 3 had 0.5 mL of hydrogen peroxide, 0.2 mL of guaiacol, and 6.8 mL of water. Lastly tube 4 had 5 mL of hydrogen peroxide, 0.2 guaiacol, and 2.3 mL of water. After creating our enzyme and substrate concentrations we covered them with parafilm until we were ready to begin our reaction. When we were ready we uncovered the parafilm from each pair, combined them into one test tube labelled reaction, inverted the tube to mix, and covered the tube once again. We set the tubes on the test rack and ran the experiment for 3 minutes and recorded observations and took pictures of the solution’s color every 30 seconds. After the trial was over we compared our pictures and observation to the color chart.
Data:
Above is a graph showing the progression of color values of each of our solutions over the three minute trial. The x axis represents time and the y axis represents the color chart. Each line represents the different substrate concentration we used. For example, the red line shows the progression of color of the substrate concentration of 0.2mL.
Claim: After our experiment we saw that increasing the substrate concentration does not always speed up the reaction. An increase in both the enzyme and substrate concentration would allow for the enzymatic reaction to occur most efficiently.
Evidence:
Color Chart
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Tube
|
H2O2
|
0
|
30
|
60
|
90
|
120
|
150
|
180
|
1
|
1
|
2
|
4
|
5
|
6
|
8
|
9
|
10
|
2
|
0.2
|
2
|
3
|
5.5
|
8
|
9
|
10
|
10
|
3
|
0.5
|
1.5
|
3
|
5
|
6
|
7
|
8.5
|
9
|
4
|
5
|
2
|
4
|
5
|
7
|
8
|
9
|
9
|
Above is shown our data chart. Each tube is shown with the substrate concentration beside it. Under each 30 second increment the color reached by the solution is shown.
Reasoning:
As shown by the evidence Tube 2 with 0.2 hydrogen peroxide, 0.2 guaiacol, and 7.1 mL of water had the fastest reaction and reached 10 by 150 seconds. This was the fastest reaction when the substrate concentration was increased. My partner and I then decided to conduct one more trial where we increased both the substrate and enzyme concentrations. For the substrate we used 0.2 mL of guaiacol, 1 ml of hydrogen peroxide, and 6.3 mL of water. For the enzyme we used 3 mL of peroxidase and 4.5 mL of water. For this speedy reaction the solution was at 3 at 0 seconds for the color chart and reached 10 by 120 seconds. This second test confirmed that increasing the substrate concentration alone is not enough. An increase in substrate and enzyme concentration can produce fast enzymatic activity.
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