Procedure 2: Determining the effect of pH on enzymatic activity
Research Question/Hypothesis:
In procedure 2 of the enzyme lab, we were tasked with determining how pH affects enzyme activity. It is important to investigate topics such as this because it helps with understanding the way in which enzymes work. In this part of the experiment our independent variable was the pH of the liquid that was used for the enzyme solution, and the dependent variable was the reaction rate when the substrate is combined with the enzyme in the test tube. Our hypothesis was that there would not be a linear trend with the rate of the reaction, but somewhere around the middle there would be an optimal pH range of about 7.
Experimental Procedure:
For this part of the lab, the procedure that we followed was outlined for us by the instructions without much variance. On the first day of the lab we conducted a control reaction with standard amounts of each substance in order to determine the amount of time that the experiments should be conducted for. After completing our control, we decided that three minutes, with recording data every thirty seconds, would be an appropriate length for the experiment. For each trial of the experiment that we conducted, the substrate solution contained 7 mL of distilled water, 0.3 mL of hydrogen peroxide, and 0.2 mL of guaiacol for a total volume of 7.5 mL. That solution was mixed in a test tube and then covered with parafilm until we were ready to start the reaction. For the experiment we were able to conduct five different trials, with the first being a control. As stated before, each substrate solution was the same for each trial, the only difference was the pH of the enzyme solution. Trial 1 was our control in which we placed 6 mL of a solution with pH 7, and 1.5 mL of peroxidase for a total of 7.5 mL of enzyme solution. Trial 2 was conducted using 6 mL of a solution with pH 5, and 1.5 mL of peroxidase for a total of 7.5 mL of enzyme solution. Trial 3 was conducted using 6 mL of a solution with pH 8, and 1.5 mL of peroxidase for a total of 7.5 mL of enzyme solution. Trial 4 was conducted using 6 mL of a solution with pH 3, and 1.5 mL of peroxidase for a total of 7.5 mL of enzyme solution. Lastly, trial 5 was conducted using 6 mL of a solution with pH 10, and 1.5 mL of peroxidase for a total of 7.5 mL of enzyme solution. After we made each of the enzyme solutions, we covered them with parafilm similarly to the substrate solutions until we were ready to run the experiment. In order to run the experiment we would pour the substrate solution as well as one of the enzyme solutions into a third test tube, cover that with parafilm, invert it twice, then let the reaction run its course. Over the course of three minutes we recorded the color of the test tube as it corresponds to a number. A number of 0 means no evidence of a reaction, and a number of 10 means the reaction has occurred essentially to completion. The numbers in the data tables correspond to the completion percentage of the reaction. The results of our experiment are included below.
Claim:
After conducting our experiment, we were able to come to the conclusion that our hypothesis was not completely correct, but we were close. It turns out that the optimal pH for this enzyme, peroxidase, to function was at a pH of 5. In our trials, we did not denature the enzyme in any of the tests, but we did see an effect of different pH’s especially as you move further away from a pH of 5.
Evidence:
The Effect of pH on Enzyme Activity of Peroxidase Over 3 minutes (Procedure 2)
pH of solution
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0 minutes
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0.5 minutes
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1.0 minute
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1.5 minutes
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2.0 minutes
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2.5 minutes
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3.0 minutes
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7
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2
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3
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5
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6
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8
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9
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10
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5
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1
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4
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6
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8
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9
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10
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10
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8
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1
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4
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5
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6
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7
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8
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10
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3
|
1
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1
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1
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1
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2
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2
|
2
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10
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1
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1
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1
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1
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1
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2
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2
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Reasoning:
As supported by the data, a pH of 5 is the optimal environment for the enzyme peroxidase to function. We came to this conclusion because this was the environment in which the color of the solution reached a corresponding number of 10 the quickest, which denotes completion. It makes sense that, for example, at a pH of 10, as well as at a pH of 3, this enzyme had very little function because the enzyme would not be in its best state. Based on the full scope of our data, the enzyme will function in each of these environments, however the enzyme would not be able to function to its full capacity the farther you go from a pH of 5. If we were able to do more tests, we would have liked to do tests on the full pH scale to figure out if a pH of 5 really is best, since we did not have access to a pH of 4 or 6.
Procedure 3: Group Chosen Variables - Substrate Concentration
Research Question/Hypothesis:
In this part of the lab, each group was given the opportunity to design and conduct their own variation of the experiment in procedures one and two. My partner and I chose to test the effect of substrate concentration on the enzymatic activity of peroxidase. It is important to understand the effect of substrate concentration on the activity of enzymes because this has real world applications. For example, substrates have to be present within an organism for enzymes to catalyze reactions, therefore, if no reactions are being catalyzed, possible deficiencies or problems could be spotted while investigating something like this. Therefore, it is important to understand the concept of how substrate concentration affects the activity of enzymes. Before conducting our experiment, my partner and I believed that, if substrate concentration is increased, then enzyme activity would increase as a result.
Experimental Procedure:
In this part of the lab, as stated before, my partner and I varied the substrate concentration in each of our trials. First, we ran a control test using the standard amounts for both the substrate solution and the enzyme solution. For our control, the substrate concentration included 7 mL of distilled water, 0.3 mL of hydrogen peroxide, and 0.2 mL of guaiacol for a total of 7.5 mL of substrate solution. The enzyme solution we used contained 6 mL of distilled water, and 1.5 mL of peroxidase for a total of 7.5 mL of enzyme solution. (Note: this exact enzyme solution was used for each trial of this part of the lab) After making sure that the reaction could finish over three minutes, we began to make the solutions for each individual trial with varying amounts of substrate. For this experiment we conducted four different trials with differing amounts of hydrogen peroxide in the substrate solution. For trial 1 we used 0.2 mL of guaiacol, 1 mL of hydrogen peroxide, and 6,3 mL of distilled water for a total of 7.5 mL of substrate solution. For trial 2 we used 0.2 mL of guaiacol, 0.2 mL of hydrogen peroxide, and 7.1 mL of distilled water for a total of 7.5 mL of substrate solution. For trial 3 we used 0.2 mL of guaiacol, 0.5 mL of hydrogen peroxide, and 6.8 mL of distilled water for a total of 7.5 mL of substrate solution. Lastly, for trial 4 we used 0.2 mL of guaiacol, 5 mL of hydrogen peroxide, and 2.3 mL of distilled water for a total of 7.5 mL of substrate solution. After mixing each of these solutions, we covered the test tubes with parafilm until we were ready to conduct each trial. When it came time to conduct each trial, we combined the substrate solution with the enzyme solution, covered it with parafilm, inverted it twice, and then let the experiment carry out. Over the course of three minutes we recorded the color of the test tube as it corresponds to a number. A number of 0 means no evidence of a reaction, and a number of 10 means the reaction has occurred essentially to completion. The numbers in the data tables correspond to the completion percentage of the reaction. The results of our experiment are included below.
Claim:
After conducting our experiment, we were able to conclude that, as you increase the substrate concentration, the activity of the enzyme does not necessarily increase. After examining our data, we came to the conclusion that you can only increase the the amount of substrate so much before the enzymes are working at their fullest capacity. Therefore, in order to yield the greatest enzyme activity, a large concentration of substrates as well as enzymes would be best.
Evidence:
The Effect of Substrate Concentration on the Enzymatic Activity of Peroxidase Over Three Minutes (Procedure 3)
Trial
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0.0 minutes
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0.5 minutes
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1.0 minutes
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1.5 minutes
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2.0 minutes
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2.5 minutes
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3.0 minutes
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1
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2
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4
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5
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6
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8
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9
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10
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2
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2
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3
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5.5
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8
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9
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10
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10
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3
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1.5
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3
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5
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6
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7
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8.5
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9
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4
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2
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4
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5
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7
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8
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9
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9
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Reasoning:
As shown by our data, the quickest acting reaction was trial 2, in which there was only 0.2 mL of hydrogen peroxide. Based on that information, my partner and I conducted a test to determine whether or not the reaction would occur quicker if you also increase the concentration of the enzyme. For this additional test we used a substrate solution that included 0.2 mL of guaiacol, 1 mL of hydrogen peroxide, and 6.3 mL of distilled water and an enzyme solution that included 3 mL of peroxidase and 4.5 mL of distilled water. After letting this experiment run, we were able to conclude that this experiment was the best of all. After just over 1.5 minutes, the reaction was already at a color of 10. Based on the combination of this final test with the data from our lab, we were able to conclude that just increasing the substrate concentration does not result in an increase of enzymatic activity. Instead, also increasing the enzyme concentration along with substrate concentration yields the best result.
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