Journal of Biological Education

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Tried and true but something new: analyzing the

enzymatic activity of catalase

Sandra M. Latourelle , Nancy L. Elwess & Amy B. Ryan

To cite this article: Sandra M. Latourelle , Nancy L. Elwess & Amy B. Ryan (2020) Tried and true
but something new: analyzing the enzymatic activity of catalase, Journal of Biological Education,
54:5, 540-547, DOI: 10.1080/00219266.2019.1620314

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JOURNAL OF BIOLOGICAL EDUCATION
2020, VOL. 54, NO. 5, 540–547
https://doi.org/10.1080/00219266.2019.1620314

Tried and true but something new: analyzing the enzymatic

activity of catalase
Sandra M. Latourelle, Nancy L. Elwess and Amy B. Ryan
Department of Biological Sciences, State University of New York, Plattsburgh, NY, USA

ABSTRACT KEYWORDS
Enzymatic activity is at the core of biological reactions. Understanding Catalase; reaction rates; pH;
them and the chemical and environmental factors that affect reaction enzymatic reactions;
rates is critical for biology students. The laboratory experiment proposed laboratory exercise
here investigated these concepts, while requiring students to document,
graph data, analyse results using statistics, and effectively communicate
results in writing. This assay is a redesign of another experiment, generat-
ing new protocols using a molecular approach, which greatly reduced the
volume of reagents needed, while reinforcing pipetting skills and mathe-
matical calculations for making solutions. Length and rigour were
increased by adding an additional two weeks for the investigation of
more catalase sources, thus more opportunity for thorough analysis of
data. Student learning was measured using different types of assessment:
pre-lab assignment, laboratory notebook checks, lab reports (individual
sections as well as the complete document), and a laboratory skills practi-
cum. A post-laboratory survey was administered to assess student percep-
tions on learning and skills acquisition. Average scores indicated success in
achieving learning objectives varied between 74.4% and 89.56%. Survey
results indicated that students had a positive impression of the activity and
felt it helped improve the skills described in the student learning
outcomes.

Introduction
In high school, students learn that enzymes have specificity, optimal environmental conditions and
play important roles in metabolic pathways. In college, these topics were revisited in more depth.
A by-product of numerous cellular reactions is hydrogen peroxide (H2O2), exceedingly toxic to
living cells (Alfonso-Prieto et al. 2009).
Some human immune system cells use H2O2 to kill foreign invaders (Paddock 2009). However,
when hydrogen peroxide is within the organelle peroxisomes, it is harmless. Peroxisomes have the
enzyme, catalase, which decomposes hydrogen peroxide, converting it to water or oxidises it to
other organic compounds, which are harmless (Cooper 2000; Lohner 2016) (Figure 1).
Catalase is an enzyme located in virtually all aerobic organisms and performs as follows:
2H2 O2 þ catalase ! 2H2 O þ O2 þ catalase
This indirect assay redesigned the original experiment (Cordts et al. 1996) by taking a molecular
approach, reducing the amounts of reagents needed (Figure 2), while introducing students to
pipetting skills and reinforcing their mathematical abilities, none of which were present in the

CONTACT Nancy L. Elwess elwessnl@plattsburgh.edu

Supplemental data for this article can be accessed here.
© 2019 Royal Society of Biology
 JOURNAL OF BIOLOGICAL EDUCATION 541

Figure 1. An overview of the catalase enzymatic reaction, showing the breakdown of the substrate H2O2 into the harmless by
products of H2O and O2. Image was generated by Sophie Kim.

Figure 2. A comparison of the amount of enzyme needed from the old method (Cordts et al. 1996) versus the updated version.
The old method needed 40 mL for each enzyme concentration, while the new method required only 400 µl for each of the
enzyme concentrations.

original experiment. In both the traditional and new approach, a potato was used initially as the
source of the catalase enzyme. This new approach also added an additional two weeks. Week one
somewhat repeated the original experiment (Cordts et al. 1996), the difference was the original
experiment broke the class into five different teams for enzyme concentrations and six different
teams for substrate concentrations. Each team was assigned only one concentration to test (in
triplicate), while this new protocol required each team to test five different enzyme concentrations,
six different substrate concentrations (in triplicates) and different pHs (Figure 3). With the original
protocol, results from the entire class were needed to accomplish what this new protocol achieved
with one team. With our new protocol, each team had ownership of all the results. Additionally, the
original protocol needed 100 x more enzyme/substrate concentrations than this protocol (Figure 2).
This is where the original experiment ended, with the combined results from 11 student teams. The

Figure 3. Sample student-generated pH experiment. The source of the catalase for this reaction was an onion. The height of
the foam at each pH was measured and recorded.
542 S. M. LATOURELLE ET AL.

new approach gave each team the freedom to research and decide what catalase source to use when
the experiment was repeated during week two, examples included bananas, green peppers, water-
melon, fresh liver, and avocado (no acidic fruits should be used). Students, based on their research,
hypothesised which catalase source, potato or their new source, would have a faster reaction rate.
Finally, week three was for students to analyse and graph all data, directly comparing the reaction
rates of the two catalase sources to draw their conclusions and determine if their results were
statistically significant (Figure 4–6). Table 1 reviews major differences between the original experi-
mental approach and this new experimental approach adopted in this paper.
According to Microtek (2018), there are substantial benefits from hands-on learning: increased
retention, increased engagement, critique opportunities, skills mastery, benefits to different learning
styles and improved problem-solving. ACS Position Statement, 2018 indicates research supports
engagement of students in well-planned laboratory experiences help foster problem-solving and

Figure 4. Sample student data indicate enzyme reaction rates comparing the results between potatoes and broccoli. The
results demonstrated that potatoes had faster reaction rates at all enzyme concentrations except 60% and 100%. Each
datapoint was done in triplicate.

Figure 5. Sample student data shows hydrogen peroxide reaction rates comparing the results between potatoes and onion.
The results demonstrated that potatoes had faster reaction rates at all enzyme concentrations. Each datapoint was done in
triplicate.

Figure 6. Sample student data illustrate catalase reactions under varying pH conditions.
 JOURNAL OF BIOLOGICAL EDUCATION 543

Table 1. Differences between experimental approaches old and new.

Categories
Targeted Group
Length of Experiment
Involvement of students in
experiment during week
one.
Involvement of students
during week two
Involvement of students
during week three
Post experiment
Amount of enzyme/substrate
needed for each
concentration
Amount of H2O2 needed for
one concentration done in
triplicate
Pipetting Skills
Math Skills
Old Approach (Cordts et al. 1996)
High school- Honors, or Advanced
Placement Biology
One lab session – 45 minutes
Each team of students did one
concentration (in triplicate)
Experiment ended with one lab
session
Experiment ended with one lab
session
Experiment ended with one lab
session
40 mL
90 mL
No pipetting was done
Table with all the volumes for each
concentration were provided for
each team.
New Approach (this paper)
College freshmen general biology
3- three hour lab sessions
(3 weeks)
Each team of students did 11 concentrations (in
triplicate)
Each team repeated the experiment using catalase
source of their choice.
Each student graphed and analyzed results to
determine statistical significance.
Students used the results and data to write their
formal laboratory reports.
400 µl
30 mL
Each team pipetted 55+ times, plus took a required
skills test on pipetting.
Teams calculated the volumes for each
concentration using C1V1 = C2V2; plus completed
a skills test on calculating solutions.

critical thinking skills, along with exposures to reactions, materials and laboratory equipment. Daniel
(2010) suggests successfully completing a well-designed project provides students with a sense of
accomplishment, as well as practice opportunities in using unfamiliar equipment. Certainly enhanced
laboratory activity regarding enzyme catalysis of catalase allowed for multiple opportunities in hands-
on and minds-on learning.
Teams addressed the following questions:

● Will more enzyme complete the reaction faster?

● What will be the effect of substrate concentration on hydrolysis?
● What pH environment will enhance or retard catalase reactions?

Intended audience and prerequisite student knowledge

The three-week laboratory activity (~ 24 students/lab; ~ 4 students per group) was part of a fifteen-
week semester freshmen level general biology laboratory curriculum designed for biology majors.
Students should have had a fundamental understanding of the scientific method and a basic compre-
hension of the preparations of solutions using C1V1 = C2V2, where Initial Concentration (C1) times
Initial Volume (V1) = Final Concentration (C2) times Final Volume (V2). Using this formula, any
variable can be determined by knowing the other three variables.

Learning time and student learning objectives

This laboratory activity lasted three weeks meeting once a week for three hours. Student learning
objectives were:
Students would:

(1) Analyse the properties of an enzyme and how various chemical and environmental
conditions influence enzymatic activity.
544 S. M. LATOURELLE ET AL.

(2) Perform mathematical calculations and make solutions using micropipettes and serological
pipets.
(3) Accurately collect, document, graph data and statistically evaluate results.
(4) Effectively communicate research results in writing.

Numerous assessments evaluated student learning: pre-lab assignment, laboratory notebook

evaluation, written assignments covering parts of the formal laboratory report, laboratory skills
assessment, and a formal laboratory report. Finally, a post-activity survey concluded the evalua-
tion process.

Methods
Materials and students instructions
A complete and detailed list of all the required materials and protocols for this laboratory activity
were provided for the students in the required and instructor-generated laboratory manual. The
redesigned catalase laboratory activity, as described in the introduction section, is provided in
Appendix 2.

Faculty instructions
Project description
A detailed outline for this laboratory activity is provided in Appendix 1. Briefly, students were
provided with supplies of catalase, phosphate buffer, 1% and 3% hydrogen peroxide, and four
different pH solutions (pH 4, 7, 9 and 11). Each group of four students explored the effect of
varying enzyme concentrations and/or varying substrate concentrations had on reaction rates,
while also determining how different pH solutions influenced the reaction (Figure 3).

Lab preparation
Appendix 1 provides thorough faculty instructions, which include a week by week timeline. Also
included in Appendix 3 is a Pre Lab assignment given to the students.

Outcomes and issues for discussion with students

This experiment was an ideal place to discuss that, as in any authentic research experiments, things do
not always go according to plan; expected results are not always guaranteed. It was possible that
mistakes could have been made through calculation errors, pipetting errors, and/or preparation of the
varying concentrations of catalase (enzyme) and/or H2O2 (substrate) solutions. This was also a good
time to reinforce the importance of keeping a good laboratory notebook, recording all the steps taken
while working through the protocol and noting any area where mistakes might have been made.

Sample data
For this three-week activity, different types of assessment/evaluation were used to gauge student
learning.
This included pre and post surveys (Table 2, Appendix 4); quizzes, grading of laboratory
notebooks, pre-lab assignment (Appendix 3), class assignments (Table 3), and final lab report
(Table 3). Since many freshmen students had never written a formal laboratory report, the class
assignments included the students completing various components of a formal laboratory report,
such as the introduction, methods, and results (all as separate assignments). Students were given
graded feedback on these assignments, and from this, the students made edits and adjustments
 JOURNAL OF BIOLOGICAL EDUCATION 545

Table 2. Survey results on the catalase laboratory activity (Scale 1–5) Appendix 4.
Survey Statement (N = 71) Average Scores
1. This laboratory activity helped me improve or reinforce my ability to perform mathematical calculations. 4.21
2. This laboratory activity helped me improve or reinforce my pipetting skills. 4.54
3. This laboratory activity helped me improve my ability to make solutions. 4.33
4. This laboratory activity helped me improve my ability to graph data. 4.72
5. This laboratory activity helped me improve my ability to analyse data. 4.11
6. This laboratory activity should be kept in the Bio 101 laboratory curriculum. 4.18
Overall Average Score 4.35

Table 3. Student learning objectives and methods of assessments, N = 163 students.

Average
Learning Objectives Assessment Method Scores
1. Analyze the properties of an enzyme and how various Graded Introductions (in preparation for their Formal a. 74.4%
chemical and environmental conditions influence Lab Reports).
enzymatic activity
2. Perform mathematical calculations and make solutions Laboratory Skills Tests a. 84.78%
using micropipettes and serological pipets. a. Pipet skills b. 89.56%
b. Math skill: Solution and dilutions & calculations. c. 82.74%
c. Pre Lab Assignment (Appendix 3)
3. Accurately collect, document, graph data, and a. Student laboratory notebooks graded a. 86.95%
statistically evaluate results. b. Graphs and analysis from each student were b. 80.17%
graded (Results).
4. Effectively communicate research results in writing. a. Methods Sections a. 76.2%
b. Student Formal Lab reports graded b. 87.82%

towards their final laboratory report. Additionally, it was felt that the laboratory skills from the
activity were equally important to measure, so the students also had to complete a laboratory skills
assessment. Students had to demonstrate correct pipetting by selecting the correct pipet, setting it
properly, picking the appropriate pipet tip and then pipetting the correct amount of water.
Included in the laboratory skills set was assessments of the students’ abilities to calculate solutions
and dilutions. For this, students were given a series of problems to calculate final dilutions.
Finally, the students were given data from which graphs were generated (using Microsoft Excel),
illustrating standard error bars and explaining the meaning of results (Figure 4–6).

Safety issues
All chemicals in this laboratory activity were no risk to low risk as a hazardous agent. The two
solutions of any concern were the pH 11 and the 3% hydrogen peroxide solutions. As a result,
students were provided with protective gloves and safety goggles to wear. All chemicals and solutions
were discarded according to our university’s biohazard waste disposal protocols and guidelines.

Results and discussion

Field testing
After the revision of the original protocol, the proof-of-concept was tested in a Bio 101 freshman
level general biology course as initially a two-week experiment. Based on the success of this
revised protocol, we expanded to three weeks to include a more in-depth analysis of results. With
this additional week, instructors were able to further evaluate students’ results for the laboratory
experiment through the formal laboratory reports. The data presented in the assessment were
based on combining results from 163 students from seven lab sections over two semesters. There
were ~ four students per lab group, and six lab groups per laboratory section.
546 S. M. LATOURELLE ET AL.

Multiple methods to evaluate student learning were done throughout this three-week activity
(Table 3). The data in Table 3 indicate the average scores for eight different assessment methods that
were used to determine if our stated Learning Objectives were met. The students’ average scores (N =
163 students), using these assessment approaches, ranged from 74.4% to 89.56% (Table 3).
Historically, it had been found that freshmen have difficulty in writing, with formal laboratory
reports being one of the most challenging. As a result, the formal lab report was broken into graded
sections (introduction with references, methods and results) prior to submission of the final product.
This way graded feedback and comments were made to allow for necessary corrections. A discussion
section was not included as part of the individual-graded sections, but it was included in the formal
lab report. The lowest scores in the assessment were in the students’ writing of the introductions
(74.4%) and methods sections (76.2%) (Table 3). In writing of their methods sections, the students
had a hard time writing about something already completed (past tense) in paragraph form and not
listed in steps. The end of the activity’s formal lab reports (due twoweeks after completion of the
activity) averaged 87.82%, far higher than the average of the individual components (introductions,
methods and results), which had an overall average of 76.92%. As a department, instructors strive to
have students average a C or better (73%). For all eight assignments, students surpassed this score.
Finally, a simple six statement survey was given to the students (Table 2, N = 71 students) to
provide feedback for this activity. Each statement was based on a 1–5 scale, with five being the
highest. The overall average was 4.35. For the statement ‘This laboratory activity helped me improve
my ability to graph data’, the survey score was 4.72 (the highest). The lowest score for this survey was
in response to the statement ‘This laboratory activity helped me improve my ability to analyse data’,
with a score of 4.11. This is a little ironic; the highest survey score was in how the activity helped in
learning to graph data, but many still struggled with how to analyse that data, which can also be seen
in Table 3 (Graphs and analyses from each student were graded) with the averages being 80.17%.

Possible modifications
In order to reinforce proper experimental design and scientific methodology, negative controls
can be included in the experiment. For the investigation of enzyme concentration on reaction rate,
the students can pipet 30 μL of water and/or denatured catalase extracted from a cooked potato on
the disk. For the investigation of substrate concentration on reaction rate, the students can pipet
10 mL of water into the glass vial rather than hydrogen peroxide. Another possible modification
or experimental extension is to investigate the effect of temperature on enzymatic activity. This
can be accomplished by conducting an experiment similar to that described in Part III, except pH
is held constant, and instead, each tube is incubated in a different temperature condition.

Other catalase laboratory experiments

Over the years, there has been a variety of catalase laboratory experiments done within the classroom
(Faure, D; Nemes R.; Osborne, J; Williamson, B. 2016). One uses potato, apple and yeast as the source
of catalase (biologycorner.com). Another lab, use potatoes but changes the temperatures at which the
reaction occurs (Enzyme Action, 2019). With both of these experiments the students were asked to
estimate the rate of reaction between the three, using a system of 0 = no reaction to 5 = very fast Our
experiment is different because we offer the students the freedom to not only select addition sources to
test, but they were also responsible for all the data analysis, which includes the timing and determina-
tion of reaction rates. Finally, in Kimbrough, Magoun, and Langfur (1997), their publication suggests
other catalase sources that can be used, but they only provided data for catalase activity under varying
temperatures.
 JOURNAL OF BIOLOGICAL EDUCATION 547

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Funding for this laboratory activity came from the Department of Biological Sciences budget for each individual
course.

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