IB Biology HL: Internal Assessment Lab Format
Personal Engagement
Catalase is an enzyme that is present in just about every living organism, and is essential for the breakdown of hydrogen peroxide into hydrogen and oxygen. Hydrogen peroxide is a substrate that is produced from cell metabolism, and although it’s beneficial in terms of helping the body fight infections, it is extremely harmful when it remains in the body for reasons other than helping to destroy pathogens. Catalase helps to prevent many harmful substances from damaging the body, and is therefore crucial for the human body. Through the examination of malfunctioning catalase, it was determined that non-functional catalase can cause increased susceptibility to apoptosis, inflammation, stimulation of tumors, and accelerated aging coupled with mutagenesis (Chelikani, Prashen, 2005).
Defining the Problem
Enzymes are molecules in the body that increase the speed of reactions. They help carry out chemical reactions that are necessary for sustaining life.
Enzymes must be able to catalyze several reactions at once. They can only bind to a specific substrate in order to initiate the chemical reaction.
The rate at which the enzyme work depends on several factors, one being substrate and enzyme concentration.
The enzyme in this experiment is called catalase; it is involved in the process of breaking down hydrogen peroxide. Hydrogen peroxide is toxic to the body and has the ability to kill cells in the absence of catalase (Lisanti, 2011).
Hydrogen peroxide is the substrate for catalase, so it is important to determine the optimal concentration of hydrogen peroxide for these reactions to occur in the human body.
Research Question
Enzymatic reactions: Investigating the effect of different substrate concentrations on the rate of the decomposition reaction of hydrogen peroxide.
Hypothesis
The rate of reaction will be the quickest at the highest concentration of hydrogen peroxide, 30ml, as increasing substrate concentration (hydrogen peroxide) will allow for more active sites for the enzyme (catalase) to bind to.
Initially, when the substrate concentration is increased, the rate of reaction increases considerably (Robinson, P. K. 2015).
Independent & Dependent Variables in the Experiment
Type of Variable
Variable
Units
Uncertainties
Independent Variable
Concentration of hydrogen peroxide
Milliliters (mL)
+/- 0.1 (10 ml graduated cylinder)
+/- 0.5 (50 ml graduated cylinder)
Dependent Variable
Rate of enzymatic reaction, as measured by the rate of the filter paper moving from the bottom of the medicine cup to the top.
cm/second
N/A
Controlled Variables
Units
Possible effect(s) on results
Method for Control
Temperature
Celsius
A higher temperature could have increased the rate of reaction.
All trials were conducted under room temperature.
Amount of time soaked in catalase
Seconds
A differing amount of catalase may have affected the rate of reaction.
Each disc was soaked in catalase for 5 seconds to ensure consistent enzyme concentration
Volume in each medicine cup
Milliliters (mL)
More or less volume could result in inconsistent rate of reaction and longer distance for rate of reaction calculations.
Each cup had a total volume of 30mL. Each filter paper traveled the same distance.
Protocol Diagram
Photograph of Lab Setup
Materials
Hydrogen Peroxide
Catalase
Medicine cups (15)
Water
Graduated cylinders (10ml and 50ml)
Stopwatch
Filter Paper
Hole puncher
Ruler
Procedure
Goggles were worn to prevent any liquids from coming in contact with the eyes. Close toed shoes were worn for protection in case the liquid spilled.
The temperature of the room was set to a consistent temperature throughout the experiment.
15 medicine cups were set up in 5 rows of 3. Each row represented a different trial.
A hole punch was used to make holes in the filter paper.
Water, hydrogen peroxide, and catalase were obtained. The proper amount of hydrogen peroxide (independent variable) and water was poured into each medicine cup, as shown below. The values for hydrogen peroxide and water were measured using a graduated cylinder. Each combination of water and hydrogen peroxide totaled 30mL in each cup.
Trial Number (Each trial had 3 medicine cups)
Hydrogen Peroxide
Water
Trial 1
0.0 (N/A)
30.0 (50 ml graduated cylinder)
Trial 2
6.0 (10 ml graduated cylinder)
24.0 (50 ml graduated cylinder)
Trial 3
12.0 (50 ml graduated cylinder)
18.0 (50 ml graduated cylinder)
Trial 4
24.0 (50 ml graduated cylinder)
6.0 (10 ml graduated cylinder)
Trial 5
30.0 (50 ml graduated cylinder)
0.0 (N/A)
Forceps were used to take a singular filter paper disc. The disc was dipped in catalase for five seconds, serving as a control.
The filter paper circle was placed on the top of each solution. Once the filter paper hit the bottom of the medicine cup, the stopwatch was started.
The time, in seconds, it took the filter paper to reach the surface was recorded using a stopwatch.
The height of the medicine cup filled to 30mL, or the distance the filter paper traveled, was measured in centimeters using a ruler to be used in the calculation for rate of reaction.
The distance over time (cm/s) was calculated to determine the overall rate of reaction.
Three replicates were performed for each trial to ensure reliable results.
Data Collection
Raw Data Table
Table 1.1: Time of Rise and Distance Traveled of Filter Discs in Various Water and Hydrogen Peroxide Concentrations
Trial
Amount of Hydrogen Peroxide Added (mL)
+/- 0.1 (10ml)
+/- 0.5 (50ml)
Amount of Water Added (mL)
+/- 0.1 (10ml)
+/- 0.5 (50ml)
Percentage of Hydrogen Peroxide
Time it Took the Filter Paper to Rise (sec)
+/- 0.0005 sec
Distance Traveled (cm)
+/- 0.05 cm
1
0.0
30.0
0%
N/A
N/A
1
0.0
30.0
0%
N/A
N/A
1
0.0
30.0
0%
N/A
N/A
2
6.0
24.0
20%
7.87
3.0
2
6.0
24.0
20%
6.98
3.0
2
6.0
24.0
20%
7.23
3.0
3
12.0
18.0
67%
4.98
3.0
3
12.0
18.0
67%
5.04
3.0
3
12.0
18.0
67%
6.34
3.0
4
24.0
6.0
80%
4.04
3.0
4
24.0
6.0
80%
3.92
3.0
4
24.0
6.0
80%
2.16
3.0
5
30.0
0.0
100%
2.06
3.0
5
30.0
0.0
100%
1.87
3.0
5
30.0
0.0
100%
1.59
3.0
*Uncertainties were calculated by dividing the smallest increment of the equipment by 2
Qualitative Data
After each mix of hydrogen peroxide and water was obtained, they appeared clear
Before the filter disc began to rise to the top of the medicine cup, bubbles accumulated on its surface while it was at the bottom of the cup
Processed Data and Calculations
Overview
After calculating the amount of time it took for each filter disc to rise throughout the trials, the average for each trial was determined to provide a concise number that accurately represented the time each disc took to rise from the bottom to the top. Similarly, the rate of reaction was calculated using the distance the disc traveled divided by the time. Once the averages were calculated, the data was presented in a line graph to effectively show the relationship between substrate concentration and average rate of reaction. Standard deviation was plotted on the graph in the form of error bars to show variability from the average rate of reaction.
Sample Calculation
Trial 5 Calculation
Average of Trial 5 Replicates (3) – (1.46 + 1.60 + 1.89) / 3 = 1.65
Data
Data – Average
(Data – Average)2
1.46
1.46-1.65 = -0.19
0.0361
1.60
1.60-1.65 = -0.05
0.0025
1.89
1.89-1.65 = 0.24
0.0576
*add numbers in (Data – Average)2 to obtain 0.0962
Table 2.1: The Average Time of Rise and Rate of Reaction of Filter Discs in Various Concentrations of Water and Hydrogen Peroxide with Standard Deviation
Trial
Average Time (s)
+/- 0.0005 sec
Rate of Reaction (cm/s)
+/- 0.0005
Average Rate of Reaction (cm/s)
+/- 0.0005
Standard Deviation
1
N/A
N/A
N/A
N/A
1
N/A
1
N/A
2
7.36
0.38
0.41
0.026
2
0.43
2
0.41
3
5.45
0.60
0.56
0.075
3
0.60
3
0.47
4
3.37
0.74
0.97
0.367
4
0.77
4
1.39
5
1.84
1.46
1.65
0.219
5
1.60
5
1.89
Conclusion and Evaluation
Conclusion
The topic that was investigated throughout experimentation was enzymatic reactions: the effect of substrate concentration on the rate of the decomposition reaction of hydrogen peroxide. It was predicted that a higher substrate concentration, or higher concentration of hydrogen peroxide would lead to a higher rate of reaction, meaning a greater number measured in cm/s. The results obtained from this experiment agreed with the hypothesis since a concentration of 100% hydrogen peroxide had an average reaction rate of 1.65 cm/s, while a concentration of 0% had no reaction occur at all since hydrogen peroxide is needed for reaction. Subsequently, a concentration of 20% hydrogen peroxide had an average reaction rate of 0.41 cm/s, showing a much lower reaction rate when compared with a concentration of 100% which had a higher reaction rate. Since there was a higher substrate concentration, the likelihood that the substrate would bind to the active site increased, since enzymes and substrates often face difficulty in binding due to the need for accuracy, direction, and timing.
The data shown on the graph further supports the hypothesis since it visualizes the effect of the increase of substrate concentration on average reaction rate. The data closely resembled a linear function, exhibiting a positive correlation. Although most substrate concentration / reaction rate graphs plateau, our graph didn’t, showing that there was no point where all of the active sites were occupied.
The standard deviation for a 20% concentration was low, meaning the data was clustered close to the mean, making it precise. Using higher concentrations, however, such as 75%, the standard deviation was larger, meaning the results were less precise.
The data presented no unusual results or anything that contradicted the hypothesis. In terms of further study, a noncompetitive or competitive inhibitor could be added to the medicine cups to determine up to what concentration the hydrogen peroxide remains effective when an inhibitor is introduced. Also, other enzymes and substrates can be used to support the trend that as substrate concentration increases, rate of reaction increases.
Evaluating the Procedure
The filter disc method was efficient in producing quick results, however the results weren’t precise throughout, shown by the high standard deviation from higher substrate concentrations such as 80%. Using another method of data collection such as the Data Logger would provide more accurate results since it wouldn’t involve errors such as starting and stopping the stopwatch and misjudging when the disc hits the bottom or comes to the top. However, the disadvantage with the Data Logger is that the corks that maintain the pressure inside the test tubes pop off, leading to irregular lines on the graph.
The only problem encountered during the experiment was that we stirred some of the cups containing water and hydrogen peroxide. Although this didn’t pose a major error in the experiment, it may have resulted in the rate of reaction being affected since the stirring may have initiated or prevented enzyme-substrate binding.
Improving the Investigation
In order to improve the investigation, Data Logger could be used for the experiment rather than the filter disc method, and would serve as a viable comparison to the filter disc method to see if it did lack precision. Another minor improvement could be to either stir or not stir each of the mixtures in the medicine cups to make the procedure more consistent. This would eliminate any variation between trials and replicates.
As previously stated, using Data Logger may provide for a more precise experiment, however the results obtained from the filter disc method showed data that supported our hypothesis. Since we only manipulated substrate concentration, all other variables were controlled throughout experimentation. Aside from the experiment we conducted in particular, other experiments using different enzymes and substrates can be performed to determine the reliability of our results. By choosing an enzyme inside the human body, effects and results from experimentation can be utilized rather than the experiment just being performed to affirm or deny a generalization such as the fact that substrate concentration has an effect on rate of reaction. Finally, other factors such as temperature and pH can be measured to determine other substrate and rate of reaction relationships.
References
Robinson, P. K. (2015). Enzymes: Principles and biotechnological applications. Essays In
Biochemistry, 59(0), 1-41. doi:10.1042/bse0590001
Chelikani, P., Ramana, T., & Radhakrishnan, T. M. (2005). Catalase: A repertoire of unusual features. Indian Journal of Clinical Biochemistry, 20(2), 131-135. doi:10.1007/bf02867412
Lisanti, M. P., Martinez-Outschoorn, U. E., Lin, Z., Pavlides, S., Whitaker-Menezes, D., Pestell, R. G., . . . Sotgia, F. (2011). Hydrogen peroxide fuels aging, inflammation, cancer metabolism and metastasis. Cell Cycle, 10(15), 2440-2449. doi:10.4161/cc.10.15.16870