Primary research on the level of potential of hydrogen buffer solution (no units) affects the dependent variable of the experiment- the volume of oxygen gas produced in a given period of time (cm3/min) and ultimately the rate of the enzymatic reaction when the substrate-hydrogen peroxide (cm3) is added?
Research Question - How does the independent variable of the experiment - the level of potential of hydrogen buffer solution (no units) affect the dependent variable of the experiment- the volume of oxygen gas produced in a given period of time (cm3/min) and ultimately the rate of the enzymatic reaction when the substrate-hydrogen peroxide (cm3) is added?
Aim -The aim of the ‘Enzymatic Decomposition of Hydrogen Peroxide by Catalase Investigation’ is to investigate how the independent variable of the experiment - the level of potential of hydrogen buffer solution (no units) affects the dependent variable of the experiment- the change in volume of oxygen gas produced in a given period of time (cm3/min) and ultimately the rate of the enzymatic reaction when a fixed amount of hydrogen peroxide is added. The aim of this experiment will be achieved by using different levels of the potential of hydrogen buffer solutions, specifically - pH 1, pH 3, pH 5, pH 7, and pH 9, all three types of solutions on the pH scale are being used – acidic solutions- the potential of hydrogen 1,3 and 5 buffer solutions, neutral solution – the potential of hydrogen 7 buffer solution and an alkaline solution – the potential of hydrogen 9 buffer solution. This will help us identify the true relationship between the acidity, neutrality, and alkalinity of a potential hydrogen buffer solution and the volume of oxygen gas produced which will help us to ultimately understand the celery catalase’s optimum potential of hydrogen level, working range, and the potential level of hydrogen at which the enzyme denatures the fastest (understand the rate of the enzymatic reaction).
Hypothesis
In this chemical reaction, the hydrogen peroxide solution (cm3) is the reactant and substrate, a substrate “is a molecule acted upon by the enzyme” (BD Editors, para 3)
The biological catalyst/ enzyme being used is celery catalase. The by-product of this reaction is water and oxygen gas. An enzyme has a unique 3D shape, therefore the shape of the active site will also be different. Only one specific substrate fits into the active site of an enzyme (this is called substrate specificity) to form an enzyme-substrate complex. Extreme temperatures and extreme potential of hydrogen levels (pH) disrupt the normal tertiary structure of the active site, resulting in the substrate being unable to fit into the enzyme, this means the key will no longer fit the lock as per the lock and key hypothesis, this process is referred to the denaturation of the enzyme.
It can be hypothesized that if 6cm3 of hydrogen peroxide solution is added to celery catalase (10cm3) and the potential of hydrogen 1 buffer solution(10cm3), the catalase will denature at an extremely fast rate. The scientific explanation for the following would be that “enzymes function best at an optimum pH” and “extremely low pH levels can result in a complete loss of activity for most enzymes”( (“Effects of PH”, Worthington Biochemical Corporation, para 3). According to KhanAcademy, Extremely low pH levels are acids below the pH level 7(the neutral pH) (‘Enzymes and Activation’ Khan Academy, para 4)Therefore it can be concluded that if a fixed volume of hydrogen peroxide is added to the catalase and pH 1 solution (after a fixed time period), the catalase begins to lose the ability “ to form ionic bonds between the substrate and the enzyme” due to the extreme acidity causing it to denature at an extremely fast rate(“10.7: The Effect of PH on Enzyme Kinetics”, para 2)Therefore, it can also be assumed that little to no by-product is produced after the reaction, meaning that there will be little to no volume of oxygen gas is produced (no effervescence is observed).
It can be hypothesized that if 6cm3 of hydrogen peroxide solution is added to celery catalase (10cm3)and the potential of hydrogen 3 buffer solution (10cm3), the catalase will also denature at an extremely fast rate, however, the rate of the reaction would be slower compared to that of the potential of hydrogen 1. The scientific explanation for the following would be that “extremely low pH levels can result in a complete loss of activity for most enzymes” (“Effects of PH”, Worthington Biochemical Corporation, para 3). However, considering that the potential of hydrogen 1 buffer solution has an ion concentration that is 1000 times greater than pH 3, pH 3 is less acidic compared to pH 1 and thus slightly less extreme acid, therefore it can be
hypothesized that if a fixed volume of hydrogen peroxide is added to the celery
catalase and pH 3 solution the catalase begins to lose the ability “ to form ionic bonds between the substrate and enzyme” due to the extreme acidity causing it to denature at an extremely fast rate, however, it is predicted to be a slower rate of reaction compared to the potential of hydrogen 1 buffer solution(“Effects of PH”, Worthington Biochemical Corporation, para 5) Therefore, it can also be assumed that a little by-product is produced after the reaction, meaning that there will be a small volume of oxygen gas produced (slight effervescence is expected).
It can be hypothesized that if 6cm3 of hydrogen peroxide solution is added to celery catalase (10cm3)and the potential of hydrogen 7 buffer solution (10cm3), the catalase would denature at the slowest rate compared to all the other potential hydrogen levels. The scientific reasoning for this would be the potential of hydrogen 7 is the optimum potential of hydrogen level for the catalase, the most favorable pH resulting in the maximal activity of a particular enzyme”(Biology Online, para 1). As mentioned extremely low or extremely high pH levels can result in a complete loss of activity for most enzymes” (“Effects of PH”, Worthington Biochemical Corporation, para 4). Considering that the potential of hydrogen 7 is a neutral level on the pH scale (not an extremely low or high pH) it can be hypothesized to be the enzyme’s optimal pH level. Hence, it can be assumed that at this potential of hydrogen level the enzyme will perform at maximal activity, resulting in the enzyme denaturing at the slowest rate compared to the other potential of hydrogen levels, it can also be assumed the greatest volume of oxygen gas is produced during this reaction (fast and vigorous effervescence is expected).
It can be hypothesized that if 6cm3 of hydrogen peroxide solution is added to celery catalase (10cm3)and the potential of hydrogen 5 and 9 (each) buffer solution (10cm3), the catalase would denature at a slower rate compared to the assumed optimum pH that is 7 but will denature at a faster rate compared to the potential of hydrogen 1 and 3 solutions. The scientific explanation for the following is that the “potential of hydrogen 5 isn’t as extreme compared to the acids on the left side of the scale as its closer to the neutral pH value of 7(“Acids and Bases: 8.31 - the PH Scale”, para 3)and the “potential of hydrogen 9 isn’t as extreme compared tothe alkalis on the right side of the scale as it’s closer to the neutral pH value of 7(“Acids and Bases: 8.31 - the PH Scale”, para 5). Therefore it can be assumed that pH 5 and 9 are within the working range of the catalase. Each enzyme has an optimum pH but it also has a working range of pH values at which it will still work well.” Therefore it can be concluded that if a fixed volume of hydrogen peroxide was added to the catalase and pH5 and pH 9 solution (after a fixed time period), the catalase would denature at a slow rate, it will denature at a slower rate compared to the potential of hydrogen 1 and 3 buffer solutions, however, it will debenture at a quicker rate compared to the potential of hydrogen 7 buffer solution as that is the optimal pH for the enzyme at which maximal product is produced. Therefore, it can be hypothesized that due to the slow rate of reaction a great volume of oxygen gas is produced compared to pH 1, 3, and 5 buffer solutions but less than pH 7). Therefore it is assumed that a large volume of oxygen gas is produced compared to pH 1 and 3 buffer solutions but lesser volume compared to pH 7 buffer solution (vigorous effervescence is expected)
In order to illustrate the hypothesis in a clearer manner, a predicted graph has been sketched.
Graph 1- A sketch of a predicted graph based on the research conducted in the hypothesis
The predicted graph illustrates how at an acidic/low pH level, the rate of the reaction is the lowest as the enzyme cannot function at such an extreme potential of hydrogen level. This is represented by looking at the shallowest upward curve on the graph.
The predicted graph also illustrates how the potential of hydrogen 5 and 9 are part of the enzyme’s working range as the rate of the reaction is quite high, slightly below the optimum pH’s rate of reaction because these potential of hydrogen levels are closer to the neutral pH compared to their extreme categories of acids and alkali respectively. This is represented by the steeper positively-sloped curve on the graph.
The predicted graph also illustrates how the potential of hydrogen 7 is the optimal pH because it is the least extreme (is neutral) , therefore this is the pH level at which the rate of the reaction is the highest. This is represented by the bell-shaped curve at the top of the graph.
In order to test the hypothesis, the independent variable will be manipulated and 5 different levels of the potential of hydrogen buffer solutions will be used, specifically – pH 1, pH 3, pH 5, pH 7, and pH 9. Before beginning the experiment, the room temperature will be measured. Since temperature also affects the celery catalase's rate of reaction, it is important to keep the temperature constant for all 3 trials. In 5 boiling tubes, a fixed amount of celery catalase will be added along with a fixed amount of a different potential hydrogen buffer solution. The mixture of these solutions will then be left to set for a time period of 7 minutes, a stopwatch will be used to control the time. During this time, a water trough will be set up by filling up a water bowl with ¾ ths of water, ensuring the water is also at a constant temperature for all three trials. An inverted measuring cylinder that is filled with water is held using the clamps on the clamp stand. After the 7-minute time period, pour 10cm3 of hydrogen peroxide into the celery catalase and the potential of hydrogen 1 solution. Immediately use a rubber tube with a stopper and place close the opening of the tube using the stopper and place the tube of the device into the opening of the inverted measuring cylinder and start a timer for 2 minutes. After 2 minutes, the volume of oxygen gas will be observed by looking at the decrease in the amount of water in the measuring cylinder. This will be noted down and the same step will be repeated for the other 4 constraints and 3 trials.
Variables
Independent Variable (IV) - “The independent variable is the variable that is being manipulated/changed and it is assumed to have a direct effect on the dependent variable, the variable that’s measured” (McLeod, para 1). In this experiment, the independent variable is the potential level of hydrogen buffer(pH) which is assumed to affect the volume of oxygen produced in a given period of time and ultimately the rate of the enzymatic reaction is a measure of how acidic/basic water is. The
range goes from 0 - 14, with 7 being neutral. pH of less than 7 indicates
acidity, whereas a pH of greater than 7 indicates a base. pH is really a
measure of the relative amount of free hydrogen and hydroxyl ions in the water.” (source,para 1). In this experiment 5 constraints are being used - pH 1, pH 3, pH 5, pH7, and pH 9. The following values for the constraints have been
used in order to determine how acidic, neutral, and alkaline pH buffers
affect the enzymatic reaction, pH is a dimensionless quantity and
therefore has no unit” (source, para 2).
Dependent Variable (DV) - “The dependent variable is the variable that is being tested and measured in an experiment” (McLeod, para5). In this experiment the dependent variable is the volume of oxygen gas produced in a given period of time (mL), this will be calculated by setting up a clamp stand which holds a graduated cylinder with the opening of the cylinder facing downwards. Below the cylinder, a water trough of water containing a fixed temperature will be placed. Once the source of catalase and all pH constraints have been left in a boiling tube for a fixed period of time, a fixed volume of hydrogen peroxide is added to the boiling tube and then is immediately covered with the stopper and the tube is placed inside the opening of the measuring cylinder that is attached to a clamp stand. Since the
measuring cylinder is already filled to water completely the change in volume can easily be calculated by reading the scale. The volume of oxygen gas produced will be calculated in the units centimeter cube per minute.
Controlled Variables (CV)
The volume of the pH - The volume of the different levels of the potential of hydrogen will be kept constant by using a graduated cylinder which has a sensitivity of 10cm3 to precisely measure the volume of each level of potential of hydrogen for all 5 constraints for all 3 trials. The volume of the potential of hydrogen needs to be controlled in order to prove that a particular volume of oxygen gas was produced due to the level of the pH buffer and not the lowered or additional volume/amount of solution that was added to each constraint.
The concentration of hydrogen peroxide -The concentration of hydrogen peroxide will be kept constant by placing only one bottle of the needed concentration (6cm3) of hydrogen peroxide in order to avoid confusion and ensure that the same concentration has been added to all 5 constraints in the experiment.The concentration of hydrogen peroxide has to be controlled as increasing the substrate concentration “will increase the rate of the reaction to a certain point” this will cause the experiment to be unfair as the rate of the reaction has been altered to due a different concentration of the substrate being added (AshokKumar V. Rajini, para 1)
The room and water temperature for the water trough - Temperature and the potential of hydrogen affect the enzymatic rate of enzymes, therefore the room temperature and temperature of the water should be the same for each trial. The room temperature has to be controlled as a lower temperature may decrease the rate of the reaction and a higher temperature may increase the rate of the reaction regardless of the level potential of the hydrogen buffer solution used. Therefore, ensuring that the temperature of the room and water in the water trough is constant throughout the experiment can ensure that the
the rate of the enzymatic reaction isn’t affected.
Mixing of pH buffer solutions - The different levels of the pH buffer solutions will be kept constant by using 5 different measuring cylinders for each of the different levels of the pH buffer solutions. A different pipette will also be used when transporting the solutions into the measuring cylinder to prevent the mixing of the different levels of pH buffer solutions. The pH buffer solutions have to be controlled in order to prevent the increase or decrease of a certain characteristic of the pH buffer (mixing can result to the solution becoming more or less acidic, neutral, or alkaline) since this is the independent variable of the experiment, mixing of the solutions can result in incorrect readings being gathered resulting in the answer to the research question not being answered - and new trials have to be conducted.
Cleaning out the measuring cylinder to prevent trapped oxygen bubbles from being counted - It is important to submerge the graduated cylinder in water using the tube with the stopper as there will still be some trapped oxygen bubbles, this can hinder with the final results as readings will be incorrect.
Apparatus to conduct 1 trial of the experiment
· Celery catalase - 50 cm3
· Hydrogen Peroxide with a concentration of 6cm3 - 30 cm3
· Gloves - 1 pair
· Safety goggles - 1 unit
· The potential of hydrogen 1 buffer solution - 10cm3
· The potential of hydrogen 3 buffer solution - 10cm3
· The potential of hydrogen 5 buffer solution - 10cm3
· The potential of hydrogen 7 buffer solution - 10cm3
· The potential of hydrogen 9 buffer solution - 10cm3
· Pipette - 6 units
· Test Tube rack - 1 unit
· Analog or digital stopwatch - 1 unit
· Boiling Tubes - 5 units
· Whiteboard marker
· 10 cm3 graduated cylinder - 10 units
· Clamp stand
· 100 cm3 graduated cylinder - 1 unit
· Water through - 1 unit
· Rubber tubing attached to a delivery tube - 1 unit
Safety Precautions
Hydrogen Peroxide is corrosive and can cause irritations if exposed to the skin, and if ingested it can become “mildly irritating to the mucosal tissue and may cause vomiting and diarrhea.”(source, para 3.)Therefore it is important to wear gloves and eye goggles when handling hydrogen peroxide.If hydrogen peroxide does contact the skin or is ingested, inform an adult and ensure to wash the affected area with water and soap. Handle glassware with care.
Method to conduct one trial of the experiment
1. Place 5 boiling tubes into a test tube rack and use a whiteboard marker to label each tube with the different levels of the pH buffer solution being used - pH 1, pH 3, pH 5, pH 7, and pH 9.
2. Gather 5 graduated cylinders and use a pipette to transport the 10cm3 of the celery solution into each of the graduated cylinders.
3. Pour one graduated cylinder containing 10cm3 of celery solution into one of the test tubes, and repeat this for the other 4 test tubes.
4. Gather a new set of 5 measuring cylinders, and use a whiteboard marker to label each cylinder with the different levels of pH buffer solutions being used -pH 1, pH 3, pH 5, pH 7, and pH 9.
5. Use a pipette to pour 10cm3 of each of the different pH buffer solutions into the corresponding measuring cylinder. Ensure that 5 different pipettes are being used - one for each solution in order to keep the experiment controlled.
6. Ensure a timer is kept ready for 7 minutes, pour the different pH buffer solutions into their corresponding boiling tubes, and immediately start the timer.
7. During this time, gather a new set of 5 measuring cylinders and use a pipette to fill each cylinder with 10cm3 of hydrogen peroxide solution. Ensure that gloves are worn during this step as hydrogen peroxide is corrosive and kills living cells.
8. Set up a water trough by using a clamp stand to hold an inverted measuring cylinder, the opening of the cylinder should be placed in a water bowl filled with ¾ with room temperature water around 23 degrees Celsius. Ensure the inverted measuring cylinder should be filled completely with water in order for the experiment to work.
9. After 7 minutes, ensure that the water trough and rubber tubing attached to a delivery tube is within reach, pour 10cm3 of hydrogen peroxide into the boiling tube containing the potential of hydrogen 1 buffer and celery solution.
10. Immediately use the rubber tubing attached to a delivery tube to connect the rubber stopped to the open boiling tube to prevent escape of oxygen and place the tubing into the opening of the inverted cylinder in order for the oxygen gas trapped to flow through the tube.
11. Set the timer for 2 minutes.
12. After 2 minutes, calculate the volume of oxygen gas released, in order to avoid parallax error, read the cylinder at eye level. Repeat the following steps for the other 4 potential hydrogen and celery solutions. Ensure that between changing the different levels of pH and catalase solution, the measuring cylinder is submerged in water in order to get rid of any oxygen bubbles to prevent incorrect data from being collected
Results
Table 1 – All the data in this data in table was collected by letting a fixed volume of celery (10cm3) and a fixed volume of the 5 different pH levels (10cm3) be added to the 5 different boiling tubes and were left to set for a fixed time period of 7 minutes. During this time total volume of hydrogen peroxide (substrate ) - 30cm3 was divided into 6cm3 using 5 graduated cylinders. After this 6cm3 of the hydrogen, solution was added to the
The final graph titled “The final graph illustrating the relationship between the independent – the level of the potential of hydrogen buffer solution and the dependent variable- the volume of oxygen gas produced in a given timer period and ultimately the rate of the enzymatic reaction when hydrogen peroxide is added“ has been drawn as per standard graphing rules, with the independent variable on the x-axis and the dependent variable on the y-axis, the graph has been drawn using the range of 10-90 for the divisions in order to display the data collected in the most logical and clear way as possible.
Graph 2 – The data for the
The final graph illustrates a downward bell shape as a curve of best fit starts from pH and rate of the reaction being zero, the rate of the reaction slightly increasing from pH 0 to pH 3 which can be observed by the shallow upward curve and then there is a rate of the reaction increases extremely fast from pH 3 to 5 and even more fast from pH 5 to 7 and at the fastest rate from pH 7 to 9 the following can be observed by the steepness of the curve increasing until it hits the maximal rate of the reaction at pH 9. The optimal potential of hydrogen can be identified to be pH 9 as this is the point at which the rate of the reaction is the highest. From pH 0 to pH 9 the graphs independent and dependent can be seen to be directly related as both the independent and dependent variables are increasing together as the curve of the slope curves upwards until the optimum pH level. From the optimal pH level 9 to pH 17, there is a rapid decrease in the rate of the reaction as it can be observed that the negative slope falls rapidly until pH 13 from which the reaction begins to decline and the catalase gets denatured at a fast rate. It can be observed that the rate of the reaction is inversely related as the rate of the reaction decreases rapidly as the pH value increases after passing the optimum pH level ((“Earth07: Direct, Inverse”, para 1)
The formula cannot be derived not only because the graph shows both directly and inversely related variables but also because they are not in
“Extreme potential of hydrogen levels and temperatures both high and low cause enzymes to denature at extremely fast rates” (“Factors Affecting Enzyme Action - What Happens in Cells and What Do Cells Need? - OCR Gateway - GCSE Combined Science Revision - OCR Gateway - BBC Bitesize”)Therefore in can be concluded that, when the potential of hydrogen It can be stated that if 6cm3 of hydrogen peroxide solution is added to celery catalase (10cm3) and the potential of hydrogen 1 buffer solution(10cm3), the catalase will denature at an extremely fast rate. This is because pH 1 contains a concentration of 1 million hydrogen ions and is the second most acidic on the pH scale https://www.sciencebuddies.org/science-fair-projects/references/acids-bases-the-ph-scale, due to this extreme pH, the active site of celery catalase will lose its specific three-dimensional shape causing the enzyme to be denatured at an extremely fast rate. It can also be concluded that if 6cm3 of hydrogen peroxide solution is added to celery catalase (10cm3)and the potential of hydrogen 3 buffer solution (10cm3), the catalase will also denature at an extremely fast rate, however, the rate of the reaction would be slower compared to that of the potential of hydrogen 1. This is because pH 3 still contains a concentration of 10,000 hydrogen ions and is still quite acidic on the pH scale
Evaluate the validity of the hypothesis based on the outcome of your investigation
According to online.stat.psu.edu, the best approach to evaluate hypotheses is by “ comparison comparing the hypothesis with the established facts”. It was hypothesized that during the formation of the positive upward slope on the graph, the independent and dependent variables will be directly proportional to one another until it hit the optimum potential of hydrogen level and from this point, it can be hypothesized that during the formation of the downward graph, the independent and dependent variables are inversely proportional to one another. However as stated in the graph analysis, there is no directly proportional relationship or an inversely proportional relationship as the independent and dependent variables are not increasing and then decreasing by the same proportion, therefore the true relationship between the independent variable can be described as – the potential of hydrogen level
Explain improvements to the method that would benefit the investigation
The two main improvements that would benefit this investigation pertain to better controlling the controlled variables of the experiment.
One of the control experiments which has to be better controlled next time is the volume of the 5 different levels of the potential of hydrogen buffer solutions, the wrong sensitivity of 20cm3 was used several times throughout the trails due to the lack of graduated cylinders with a sensitivity of 10cm3. This made it more difficult to measure and read the volume. Hence parallax error may have occurred even after viewing the graduated cylinder at eye level, this may have hindered the data for the volume of oxygen gas collected in a given period of time. Therefore, next time the experiment the number of units of an item required for the experiment must be prepared beforehand.
Another control that has to be better controlled next time is temperature. We forgot to measure the room temperature before conducting our 3 trials on 3 separate days and timings. Since temperature is a factor that affects the volume of oxygen gas produced in a given period of time and ultimately the rate of the enzymatic reaction. Therefore, the next time I would record the room temperature of the room before conducting the experiment and I would either conduct the experiment with artificial lights or closed curtains to keep this variable constant. It would also be beneficial to conduct more trials in order to get the truest results.
Explain extensions to the investigation that would benefit the investigation
If I had the opportunity to conduct the ‘Enzymatic Decomposition of Hydrogen Peroxide by Catalase Investigation’ again instead of investigating how the independent variable – the level of the potential of hydrogen buffer solution ( no units) affects the dependent variable of the experiment- the volume of oxygen gas produced in a given period of time when hydrogen peroxide is added. I would like to investigate how the independent variable – the potential of hydrogen affects the dependent variable of the experiment – the volume of simple sugars (released juices from the cells) when Pectin is added. In order to conduct this experiment, I will use a range of independent variables – pH 1, pH 7, and pH 14. The controlled variables for this experiment would be the volume of the pH buffer solution, the concentration of Pectinase(substrate) added, the room temperature, the duration of letting the Pectinase sit with the different levels of pH, and the duration of the pectinase sitting with the Pectin(substrate). The rate of the reaction will be measured using a dialysis tube as it’s a liquid and the process of diffusion is taking place. It can be hypothesized that the optimum pH is 7 and the pectinase will denature at an extremely fast rate as both pH 1 and pH 14 are extreme pHs on the opposite sides of the pH scale. A similar method like this investigation will be conducted in order to identify the true relationship between the independent and dependent variables.
Works Cited
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