Introduction: Enzymes are organic catalysts that speed up a chemical reaction in a cell. Due to its structure, changing the pH level of an enzyme’s cellular environment will denature the enzyme, also disrupting its function. Yeast contains the enzyme, catalase. Catalase speeds up the breakdown of hydrogen peroxide, a toxic by-product found in cells that disrupts cell activity. When hydrogen peroxide encounters catalase, it breaks down into water and oxygen.
2H2O2 (aq) H2O (l) + 2O2 (g)
Enzymes are highly sensitive to changes in their environment, and are effective and active at their temperatures and pH levels. Variations in an enzyme’s cellular environment, such as pH alterations, leads to the effect of an enzyme’s structure, which in turn denatures the protein. Enzymes bind to a substrate molecule by their complementary shapes that results in the loss of 3D structure of an enzyme. By altering the structural bonds to change in the active site’s shape, H2O2 is prevented from binding to the enzyme’s active site.
If the enzyme catalase denatures in the presence of hydrogen peroxide, then the decomposition rate of hydrogen peroxide into water and oxygen gas will be significantly slower. In this investigation, the rate of hydrogen peroxide breakdown and production of oxygen can be measured by the time in which the yeast covered disks rise to the surface of the beaker.
Hypothesis: The closer the hydrogen peroxide solution’s pH is to 7, the faster the rate of catalase activity will be.
Variables: Independent Variable- pH of hydrogen peroxide solution
Dependent Variable- rate of catalase activity
Constant factors: Temperature of yeast suspension
Concentration of hydrogen peroxide solution
Amount of yeast covering each disk
Volume of hydrogen peroxide in each trial
Circumference of the filter paper disks
Materials:
Procedure:
1. A suspension of yeast was prepared for the class by adding 10g of yeast to 100mL of warm water (300C).
2. Approximately 1.5cm of the yeast suspension was poured into a plastic medicine cup.
3. 40ml of hydrogen peroxide solution was measured in a 50ml measuring cylinder, then poured into a small beaker.
4. A hole punch was used to cut approximately 10 disks (5mm diameter) from the filter paper, whilst ensuring the disks were not touched by the students’ fingers, to avoid contamination.
5. The yeast mixture was maintained at a constant temperature of 300C in a glass beaker with warm water. The yeast temperature was measured at 300C with a thermometer.
6. A single disk of filter paper was dipped into the yeast suspension using tweezers, with the excess yeast being shaken off.
7. The yeast-covered disk was then placed into the beaker of the hydrogen peroxide solution, with various pH, dependant on each group. (eg. pH 3)
8. The time taken for the disk to rise from the bottom of the beaker to the surface was timed with a stopwatch and the resulting time recorded in seconds.
9. Each item of apparatus was thoroughly cleaned with distilled water and wiped dry using paper towel.
10. Steps 5-9 were repeated at least five times until consistent data was obtained.
11. From the results, an average time in seconds, for the discs to rise in each beaker, was determined.
12. The results from each pH group were shared with the remainder of the class, and the data, including averages, combined in a table.
13. The rate of the catalase reaction was then calculated using the reciprocal of the average time and converted to scientific notation.
Example:
(pH 3) average time taken for disks to rise = 50.5 seconds
Estimate of the rate of reaction = 1/50.5 sec-1
= 0.0198 sec-1
In scientific notation = 1.98 x 10-2 sec-1
Results:
pH
3
5
7
9
11
Trial 1
52.3
16.7
23.4
77
31.7
Trial 2
57.5
23.2
21.5
78
36.7
Trial 3
48.4
20.3
26
80
39.4
Trial 4
48.9
18.4
25.3
66
36.4
Trial 5
45.3
15.1
21.4
93
44.5
Average (sec)
50.5
18.7
23.5
78.8
37.7
Reaction Rate (sec-1)
0.0198 sec-1
0.0535 sec-1
0.0426 sec-1
0.0127 sec-1
0.0265 sec-1
Reaction Rate (scientific notation) (sec-1)
1.98 x 10-2 sec-1
5.35 x 10-2 sec-1
4.26 x 10-2 sec-1
1.27 x 10-2 sec-1
2.65 x 10-2 sec-1
Evident in the quantitative data present in figures 1 and 2, the highest rate of catalase activity was at 0.0535 sec-1 and was achieved at the pH of 5. It can be concluded that the enzyme catalase is best suited in an environment of hydrogen peroxide with a pH between 5 and 7.
Discussion: According to the practical results, catalase reached an optimum pH level of 5 when the rate of reaction reached 5. The next fastest reaction rate was achieved at pH 7. These two results are the most reliable in the investigation when compared to the remainder of the data. They also support the hypothesis; that the closer the hydrogen peroxide solution’s pH is to 7, the faster the rate of catalase activity. Although the optimum pH environment of yeast catalase is said to be between 6 and 7, the data does in some way confirm the hypothesis but not entirely, as the hypothesis was out by 2 pH levels. The data shows that the hydrogen peroxide with a pH of 9, produced the lowest measureable rate of catalase activity, as the yeast, took an average of 78.8 seconds for the hydrogen peroxide to decompose and produce oxygen and water. With reference to the results table and graph, a pattern can be identified which indicates that each trial measuring catalase activity was consistent at each pH level.
Random Errors are caused by any factor that randomly affects the measurement of a variable. In this investigation, possible random errors could include misjudgement and inconsistency of the trial’s end point. This would be caused by human judgement and inconsistent verdicts of each participant for each test. In each group responsible for a particular pH level, problems could arise when sharing the role of participation. One participant could judge the yeast covered disk as ‘reaching the surface’ as soon as the edge of the disk touches the surface, whereas another participant may determine the end point once the disk has skimmed the surface, lowered, and then finally floated to the top and stayed in position, horizontally.
This imprecision is evident in trial number 5 of the pH level 9, where the result is 14.2 seconds higher than the average interpretation for that particular pH. An improvement to this error could include keeping the leading participant constant throughout the experiment, and delivering the same instructions to each student, ensuring they are all aware of when the exact end point of the test is.
With each group having been supplied previously prepared solution of hydrogen peroxide, it was assumed that the pH level was representative of its label. No group was sure that their pH level was accurate as no testing prior to their trials, was used to confirm the pH of the test solutions prior to testing. This error could have been avoided by ensuring correct calibration of pH indicators is completed for the participants to test and approve the pH to match the pH of the solution.
Due to the use of biological species, the yeast sample for each group may have had a different metabolic rate, and rates of breaking down hydrogen peroxide into water and oxygen, although each sample was obtained from the same species. A way to improve this random error is by repeating more trials (at least 10 for each pH) to ensure reliability of results. Another possible random error could include misunderstandings of the practical’s outline and instructions. This could have resulted in obtaining incorrect data, irrelevant to the practical, or even incorrect and inconsistent measuring of the materials present in the experiment.
Also, the temperature of the hydrogen peroxide relevant to each group was most likely not a consistent temperature throughout each group as only the yeast suspension’s temperature was kept constant, whilst the hydrogen peroxide was not tested with a thermometer or kept within the same temperature as the other solutions. Testing the hydrogen peroxide with a thermometer and keeping the solution cool in an ice bath would help to ensure consistent results are obtained throughout the test. Bubbling and therefore, production of the oxygen occurred before the yeast-covered disk was at the bottom of the beaker. This caused inconsistent times of catalase activity to be recorded as the timer started once the disk hit the base of the beaker and stopped once it was situated at the surface. A way to overcome this random error is by employing the use of a gas syringe to accurately measure the exact amount of oxygen produced, and thus catalase activity, as soon as the yeast is dropped into the hydrogen peroxide solution.
Systematic Errors occur when faulty or incorrectly calibrated apparatus or experimental design, causes values to differ consistently from their true values. In experiments, it is crucial to repeat the tests to identify any systematic errors that are consistent throughout the experiment. A source of systematic error could have been the removal of excess yeast during the preparation of the disk as it wasn’t consistent throughout due to human error. As each participant has a different judgement, it is almost impossible to ensure precise and consistent amounts of catalase are present for each trial within every group. No measuring device was utilised to ensure the amount of yeast solution on each disk was consistent, causing each trial to vary in the concentration of catalase.
By cleaning the equipment after each trial with distilled water it may have potentially left behind substances from the previous trial, that may act as a contaminant. By washing the used apparatus with only distilled water, rather than detergent, the removal of contaminants wouldn’t have been correctly carried out. A dishwasher and/or detergent would come to use, or obtaining sufficient amounts of equipment, five of each piece of apparatus for each group, to ensure all equipment is free of impurities and cross-contamination. The parallax error would have been evident throughout this practical and represents another systematic error. This error occurs when the meniscus is incorrectly read and thus, incorrect measurements recorded. In relation to this experiment, when participants read the measuring cylinder containing 40mL of H2O2, it is common that not all students determined where exactly the meniscus was, due to an alteration in where the students positioned themselves when measuring. A way to overcome the parallax error is by ensuring all participants are aware that the meniscus should be at eye level to 40mL, otherwise errors in their readings will persist. An example of the correct determining of the meniscus is shown below: alternatively, the material could have been measured more accurately by heights using a calibrated scientific
balance.
Precision refers to the amount of scatter in the results, which determines how well random errors are minimised. Accuracy refers to how close the results of the experiment are to the true values. For a ‘fair test’, it is important to ensure all other factors are kept constant to minimise the effect they have on the results, so that they don’t conflict with the independent variable. By increasing sample sizes to 10 trials, far more accurate averages can be determined to help reduce the effects of random error, eliminate outliers and produce more reliable results. It is possible to detect systematic errors by repeating the experiment and comparing results to establish whether the data collated reflects the expected true values.
Safety issues are as follows:
Hazard
Operating Procedures
Chemical use- Hydrogen Peroxide
Chemical could burn skin if it comes in direct contact. Also, eyes can be effected when coming close to the chemical’s fumes. Glasses and lab coats are advised throughout the entire experiment.
Broken glass/mercury
By using glass apparatus such as beakers and thermometers, can cause danger if they are dropped. Broken glass becomes a hazard in the lab to all students and should be swept up and placed in the bin immediately after breakage.
As well as broken glass, the mercury inside the thermometer would be a huge hazard for mercury poisoning is gloves were not worn.
Spillage
Liquids can result in spillage which, like the broken glass, need to be cleaned up instantly to prevent slipping. Paper towels and mops should be used to clean up any spillages.
Allergic reaction
By foreign substances encountering the skin, they may cause allergic reactions. Any itching, swelling or redness needs to be treated with water and/or ice, and if symptoms persist, medical assistance is required.
Staining of clothes
Not wearing correct laboratory attire, such as a lab coat, can result in substances spilling on clothes and possibly staining them. It is advised to always wear a lab coat during a practical.
Boiling kettle
Students not wearing gloves when pouring water from the kettle into their beakers, could cause burns to arise if any spillage occurs.
Ventilated workplace
Using toxic chemicals such as H2O2, can result in toxic fumes/vapours/mists to be present in the air of a laboratory. Removing contaminated air by opening windows and doors, helps to ensure students aren’t exposed to a poorly ventilated workplace.
Conclusion: Through comparison of the practical results to the hypothesis predicted, it can be clearly understood that the closer the cellular environment, of the enzyme catalase, is to a neutral pH of 7, the faster the rate of catalase activity. It can be concluded and confirmed that through a series of trials, the effect of pH levels can cause changes to an enzyme’s environment, and thus, the activity rate of the enzyme catalase.