During the enzyme lab, we conducted an experiment by combining a substrate, an indicator, and an enzyme. We also included a neutral buffer to maintain the pH level at around 7. When all these components were mixed together, we observed a noticeable change in the color of the substance. The substance gradually became darker and took on a brownish hue as more oxygen was produced during the reaction. Our analysis showed that oxygen production increased by about 10% per minute until it reached equilibrium at 4 minutes. After this point, there was no further change in oxygen production until the fifth minute. If any modifications were to be made to our experiment, our aim would be to achieve more precise comparisons with our reference chart. In conclusion, we considered the lab overall successful.
In the second part of the enzyme lab experiment, there were twelve test tubes. Six of these test tubes (labeled #1, 2, 4, 9, 11, and 12) contained a substrate with guiacol as the product indicator and a neutral buffer. The other six test tubes (labeled #3, 5, 6, 7, 8, and 10) contained turnip peroxidase enzyme with different pH solutions for each tube: pH levels of 3, 5, 6, 7, ,8 and ,10 respectively. It’s important to note that as the pH decreases in liquid acidity increases while it becomes more basic or alkaline when pH increases. To conduct the experiment we mixed each of these six test tubes with one containing its respective chemicals and indicated pH level from the instructions given. We measured oxygen production using the same color palette method used in part one of this lab. Our results showed that reaction rate increased proportionally to neutrality of pH level being closer to a pH value of approximately seven.
When the pH level was extremely acidic or basic, the reaction rate decreased. In terms of color comparison, the solution appeared darker as the pH approached neutrality. Conversely, the solution appeared lighter when the reaction rate was lower. This phenomenon occurred due to the enzyme’s susceptibility to external factors. In this particular experiment, pH served as the factor, but other variables such as temperature and inhibitors could also disrupt the reaction. If we were to modify this experiment, we would test all pH levels ranging from 1 to 14 and arrange them in numerical sequence for a more accurate comparison of our data. Overall, we deemed this experiment to be reliable.
In part three of our experiment, we designed our own experiment by controlling the temperature of the enzyme. We believed that increasing the temperature of the enzyme to 100 degrees Fahrenheit would slow down the reaction rate, possibly even stopping it completely, due to the enzyme’s functional range. At higher temperatures, enzymes are known to have less reactivity with substrates.
We set up two test tubes. In one test tube, we added a neutral buffer, 0.1% H2O2 substrate, and a product indicator. In the second test tube, we added our enzyme, turnip peroxidase, along with the neutral buffer, and heated it on a hot plate. This was done to reduce the enzyme’s productivity. We then combined the two solutions and observed them every minute.
Our hypothesis proved to be correct as the reaction rate was 25% slower than expected. If we could make any improvements to this experiment, we would ensure a more accurate temperature measurement of our enzyme. Overall, this experiment successfully demonstrated the impact of temperature on enzyme activity.
Question & Hypothesis
Part 1
The objective is to determine if oxygen can be produced by combining a substrate, enzyme, and a neutral buffer. It is hypothesized that if all substrate and enzymes interact, a reaction will take place resulting in the production of oxygen as all reactions generate a product.
Part 2
Do different pH levels impact the amount of oxygen generated during the reaction? Using a neutral buffer with a pH of 7 in part 1 means that the percentage of oxygen produced would be influenced due to the varying stabilizing effects of different pH levels.
Part 3
If we increase the temperature of the enzymes to 100° Fahrenheit, the reaction rate will slow down or even stop due to exceeding its functional range.
Methods and Procedures
Part 1
1. We used a syringe to measure 2.5 mL of 0.1% H2O2 (the substrate), 2.5 mL of guaiacol (the product indicator), and 10 mL of neutral buffer (pH 7) and transferred them into test tube SPNB.
2. We then mixed the contents of the SPNB test tube by inverting it twice.
3. Using a syringe, we measured 2.5 mL of turnip peroxidase (the enzyme) and 10 mL of neutral buffer (pH 7) and transferred them into test tube ENB.
4. To ensure thorough mixing, we inverted the ENB test tube twice.
5. The contents of test tube SPNB were transferred into ENB using a disposable transfer pipet and the solution was mixed by inverting it twice.
6. We observed and compared the color changes of the reactions every minute to the color palette, repeating this process for a total of 5 minutes.
Part 2
1. In tubes 1, 2, 4, 9, 11, and 12, we combined diluted hydrogen peroxide (the substrate) – measured with a syringe – with guaiacol (the product indicator) and neutral buffer (pH 7), totaling to 2 mL of diluted hydrogen peroxide, 1 mL of guaiacol, and 1 mL of neutral buffer in each tube.
2. In tubes 3, 5, 6,7 ,8 ,and10 , using a syringe again we mixed turnip peroxidase (the enzyme) – measured as well – with neutral buffer (pH corresponding to the test tube number e.g., pH5 for test tube5). We combined them by adding1 mL of turnip peroxidase and3mLof neutral buffer to each respective tube.
3. To create a combined solution from steps one and three in all six tubes- starting fromtubeone-we mixed the solution in tube3with thatin tubel.This mixing process was repeated for all sixtubes,resulting ina totalof sixtest tubes containing thecombined solutionsfrom stepsonesandthree.
4. We observed the test tubes over a duration offive minutesat one-minute intervals,and compared the colors produced from thereactiontothecolorpaletteprovided.
Part 3
1. With a syringe, we measured and added 2.5 mL of 0.1% H2O2 (the substrate), 2.5 mL of guaiacol (the product indicator), and 10 mL of neutral buffer (pH 7) to test tube SPNB.
2. To mix the contents, we inverted the SPNB test tube twice.
3. Using a syringe, we measured 10 mL of neutral buffer (pH 7) and transferred it to test tube ENB.
4. The enzyme, turnip peroxidase, was heated to 100° Fahrenheit and placed in test tube ENB.
5. To ensure thorough mixing, the ENB test tube was inverted twice.
6. To transfer the substance from test tube SPNB to ENB, we used a disposable transfer pipet. The solution was then mixed by inverting it twice.
7. We observed and compared the color changes of the reactions to the color palette at intervals of 1 minute. This process was repeated for a total of 5 minutes.
Data: Tables and Graphs
Time – Minutes
pH – Percent
1
60%
2
70%
3
80%
4
90%
5
90%
Rate
0.75
Part 1
Part 2
Minutes
Tube #3
Tube #5
Tube #6
Tube #7
Tube #8
Tube #10
1
40%
70%
60%
60%
50%
0%
2
40%
80%
70%
70%
60%
10%
3
50%
90%
80%
80%
60%
10%
4
60%
100%
80%
80%
70%
10%
5
70%
100%
90%
90%
80%
10%
Part 3
Minutes
pH Level
1
30
2
40
3
40
4
50
5
50
Conclusion
Part 1
The objective of the study was to investigate how oxygen is produced by combining a substrate, enzyme, and neutral buffer. Our hypothesis suggested that the reaction between the substrate and enzyme would yield a product. The results demonstrate that oxygen was indeed generated at a consistent rate of 0.75 or 75% per minute. This rate steadily increased until reaching its peak at 4 minutes. The pH level remained stable at 90% from minute 4 to minute 5. To enhance the accuracy of the experiment, more precise measurements should have been taken for the substrate, enzyme, neutral buffer, and product indicator due to a minor discrepancy caused by an air bubble during syringe measurement.
Part 2
In summary, we examined how different pH levels impacted the percentage of oxygen generated during the reaction. We had a hypothesis that using a neutral buffer with a pH of 7 in part 1 would affect oxygen production due to the varying effects of different pH levels. The data confirms this idea, as pH 10 showed the lowest oxygen production (10%), followed by pH 3 (70%). The graph depicting the results resembled a hill rather than a linear trend, indicating that moving further away from pH 6 or pH 7 would lead to decreased reaction. Our belief was shaped by our understanding that a neutral buffer with a pH of 7 helps stabilize the reaction in part 3.
Our objective was to investigate the effect of enzyme temperature on reaction rate. We hypothesized that raising the temperature to 100° Fahrenheit would lead to a decrease or cessation of the reaction, as it exceeds the optimal range. The data demonstrates that increasing the temperature reduced the reaction rate and extended the time taken for pH percentages to transition compared to when the enzymes were cold. Specifically, it took 2 minutes for pH percentage to increase from 40 to 50, whereas in part 1, only a minute was required for movement to the next pH percentage. In part 2, the rate was 0.5 or 50% per minute, in contrast with part 1’s rate of 75% per minute. If we were to modify this experiment, we would use Celsius instead of Fahrenheit because converting from 100° Fahrenheit to 38° Celsius may have affected our results due to rounding and imprecise thermometer readings.
Possible Sources of Errors:
Part 1: Several errors may occur, such as inaccurate measuring of the substrate, enzyme, neutral buffer, and/or product indicator. Another potential error is not comparing the reactions to the color palette at precisely the same time and misinterpreting the comparison.
Part 2
There may have been errors during the experiment, such as putting incorrect pH levels in test tubes, inaccurate measurement of the substrate, enzyme, neutral buffer, and/or product indicator. Additionally, comparing test tube reactions at different times and making poor judgments when comparing reactions to the color palette could also have led to errors.
Part 3
Possible errors may have occurred due to:
- Imprecise measurements of the substrate, enzyme, neutral buffer, and/or product indicator
- Failure to heat the enzymes at precisely 100° Fahrenheit (as we had to convert it from Celsius to 38° Celsius)
- Premature or delayed comparison of the reactions to the color palette
- Inadequate judgment when comparing the reactions to the color palette
Part 2 Assessment Questions
1. Enzyme reaction rate is higher when the pH level is closer to neutral because enzymes maintain equilibrium within a narrow range, and any changes in pH affect them whether towards acidity or alkalinity.
2. The enzyme remains unchanged throughout the reaction while reacting with the substrate, resulting in increased oxygen production in the tube.
3. The reaction relies on all chemicals being present, and removing some of one chemical has less impact than removing all of it. If we remove the enzyme, there would be no substrate for it to react with and thus no reaction would occur. Removing the substrate means there will be nothing for the enzyme to react with, and removing the indicator prevents observation of the reaction.
4. Other factors that influence enzyme activity include temperature, substrate availability, exposure to light, and pH affects enzyme activity by promoting greater reactivity at a more neutral pH compared to acidic or alkaline levels.
5. Enzymes have a role in breaking down food in your mouth as well as waste in your intestines.
6. Mammalian peroxidase may exhibit higher activity due to mammals being more complex organisms compared to turnips.
