pubs.acs.

org/jchemeduc Laboratory Experiment

How Should Apples Be Prepared for a Fruit Salad? A Guided Inquiry

Physical Chemistry Experiment
Reneé S. Cole, Marc Muniz, Erica Harvey, Robert Sweeney, and Sally Hunnicutt*
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ABSTRACT: In this guided inquiry experiment, students extract catecholase enzyme from
apples to catalyze the oxidation of catechol. They follow the reaction using the UV−vis
absorbance of the p-benzophenone produced to determine the Michaelis−Menten kinetic
parameters. Students make selected experimental choices within a structured framework such as
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selecting the apple varietal, the pH of the reaction mixture, and the reaction inhibitor. The
experiment has been tested at multiple universities in physical chemistry laboratory courses with
both large and small enrollments. We describe the experiment and its implementation in both
synchronously and asynchronously taught courses.

KEYWORDS: Laboratory Instruction, Physical Chemistry, Inquiry-Based/Discovery Learning, Kinetics, Enzymes,

Upper-Division Undergraduate

■ INTRODUCTION
Enzyme kinetics is a topic central to the study of biological
Kinetic studies of enzyme/substrate reaction systems have
proven fruitful as subjects for a wide variety of undergraduate
systems. In a physical chemistry context, enzyme kinetics experiments. Targeted courses for these experiments include
represent a unique opportunity for students to be introduced general chemistry, physical chemistry, organic chemistry,
to the physicochemical phenomena that underpin the biochemistry, biophysical chemistry, and even middle and
functioning of enzyme−substrate systems. According to a high school science.6 The majority of the inquiry-based
recent survey of instructors to assess the state of physical experiments were project-oriented experiments that required
chemistry courses across the U.S., the topic of “rate laws” was multiple lab meetings (even up to an entire semester). For
the fifth most covered of 16 topics in thermodynamics/kinetics example, in these project-oriented experiments, the students
with 69% of respondents reporting a “great” level of coverage determine the Michaelis−Menten kinetic parameters as a part
of the topic.1 This finding indicates that the topic is one that of a larger biochemical study of the enzyme and substrate7−10
experts in physical chemistry designate as important for or as part of a comparison of enzymatic reactors.11 Different
students to explore. One approach to making this topic measurement techniques have been used to determine the
amenable to student exploration is to situate the learning concentration of products with UV−vis spectroscopy being the
experience in the context of a system that is both familiar and most common.8−10,12,13 Other experimental apparatus used
relevant to students.2,3 include ion-selective electrodes,14 pressure sensors,15 displaced
Enzymatic browning of fruits, such as apples, is mediated by volume apparatuses,6 fluorescence spectroscopy,16 and blood
the enzyme catechol oxidase. Pyrocatechol is the substrate of glucometers.17 An important feature of all these experiments is
the enzymatic reaction and is an antiseptic compound released their appeal to student interest, including the use of yeast,6
by the fruit once its outer layer is penetrated. The enzyme ethanol,9 urease,12 and the production of vanilla.7 The enzyme
interacts with pyrocatechol and oxidizes the catechol to in most reports may be purchased commercially, while in a few
benzoquinonethe compound whose electronic absorption
properties are responsible for the brown color observed in
oxidized fruit.4 This natural enzymatic browning process is Received: May 25, 2020
estimated to be responsible for up to 50% of commercial losses Revised: September 28, 2020
and therefore has significant economic impact.5 We chose the
browning of apples as a highly relevant system for students to
investigate in an inquiry-based, physical chemistry laboratory
setting.

© XXXX American Chemical Society https://dx.doi.org/10.1021/acs.jchemed.0c00517

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reports the enzyme extraction or production is part of the

experimental procedure.12,16
Physical chemistry experiments typically emphasize data
modeling and often rely on applying linear models to data. The
Michaelis−Menten mechanism yields a nonlinear relation that
may be linearized as a reciprocal−reciprocal plot. Earlier
reports of enzyme kinetics papers tend to emphasize this linear
method of data analysis (for example, see Lewis et al.15). As
spreadsheet programs or other software become more
ubiquitous, experiments reported in the Journal now use
nonlinear modeling of the Michaelis−Menten equation to
arrive at the kinetic parameters.18,19 Some experiments use
advanced modeling techniques. Barton20 reports using Eadie−
Hofstee linearization to extract kinetic parameters from the
data. Her et al.21 describe an experiment in which students
employ the Lambert-W function and progress curve analysis to
extract the Michaelis−Menten kinetic parameters from a single
experiment at one substrate concentration by monitoring the Figure 1. Learning cycle from POGIL−PCL25 (reproduced with
change in the NMR signal as a function of time. This analysis permission by the American Chemical Society).
requires tools not found in a typical spreadsheet program, so
the analysis could be a limiting factor depending on and come back to pre-experiment questions for the next part of
institutional resources. the experiment (thus repeating the cycle). Students are led to
Another aspect of reviewing existing experiments is the level apply mathematical models during the subsequent runs
of inquiry expected of students. We adopt a perspective through the same cycle. A set of postexperiment questions
inspired by the work by Bruck et al.22 along with the Next prompt students to apply concepts learned to new situations or
Generation Science Standards23 to define the inquiry levels in to delve more deeply into theoretical aspects of the current
the cited enzyme kinetics experiments. An example of a experiment. In the scheme described by Bruck et al., the
confirmation experiment is the report by Lewis et al.;15 their experiment falls into the guided level of inquiry-based
experiment provides to the student all aspects of carrying out experiments.
the experiment. Silverstein9 describes a structured or guided Most published descriptions of inquiry-based laboratory
inquiry experiment focused on alcohol dehydrogenase. In this experiments are designed and implemented in laboratory
6 week enzyme kinetic experiment, students select which environments where all students in each laboratory section
enzyme inhibitor to test, and they must determine all solution complete the experiment simultaneously. This allows for a
concentrations and volumes needed for their experiment. The distribution of experimental conditions and opportunities for
semester-long project-oriented experiment described by whole class discussion to more fully explore issues related to
Sarisky et al.8 includes some aspects of a confirmation experiment design and analysis. However, upper level
experiment in the early weeks but builds to authentic inquiry laboratory courses in many institutions are implemented
by the final weeks of the experiment. Students make and test using a rotation model where student teams rotate through
hypotheses concerning the role of certain amino acids in the the experiments (typically due to limitations in available
enzyme, and they also make all decisions related to solution instrumentation). Only one or two groups carry out a
preparation (concentrations and volumes). Notably, it is particular experiment at any given time, and multiple
possible for students to experience a research “failure” in the experiments are being conducted during the laboratory period.
project, yet all students will be able to achieve all the desired The rotation model creates challenges for implementing
experimental outcomes including the kinetic analysis. inquiry-based experiments because there are minimal oppor-
The original experiment described here was developed as tunities to compare data and because the instructor (often a
part of the Physical Chemistry On Line (PCOL) Project24 and graduate teaching assistant) monitors several different experi-
was then adapted to the pedagogical framework for active- ments simultaneously. Our goal was to ensure that the
learning in the laboratory (Process Oriented Guided Inquiry experiment described here could be modified to function in a
LearningPOGIL) for physical chemistry.25,26 The POGIL− rotation style laboratory environment as well as in the more
PCL (physical chemistry laboratory) project is an NSF-funded easily facilitated laboratory environment where all students
effort to grow and sustain a community of instructors who complete the experiment simultaneously.
develop, test, refine, and publish inquiry-based laboratory
experiments for physical chemistry.
Figure 1 illustrates the experiment cycle developed by faculty
■ EXPERIMENT OVERVIEW
Learning Outcomes
participants in the POGIL−PCL project. For any such
experiment, the title is a question; students complete the The experiment has intended learning outcomes that address
experiment in order to answer the question, rather than to both content knowledge related to enzyme kinetics and
confirm a numerical value. To investigate the first question, laboratory skills related to experimental design and data
students must proceed through the cycle in Figure 1 by analysis. Specifically, the experiment is designed to facilitate
addressing pre-experiment questions, making initial predic- students being able to do the following:
tions, carrying out the investigation in the laboratory, (1) analyze and manipulate equations and graphical
responding to “thinking about the data questions” that guide representations to appropriately model experimental
students to focus and reflect on relevant features of the data, data/results
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(a) fit a nonlinear function to data students monitor the reaction progress of three mixtures each
(b) convert a nonlinear relationship to a linear form containing catechol:
(c) extract relevant information from the results (i.e., • 2.0 mL of catechol in water with 0.5 mL of water
slope, intercept) • 2.0 mL of catechol in water with 0.5 mL of apple juice
(2) choose experimental conditions to obtain appropriate extract (the enzyme)
data for analysis • 2.0 mL of catechol in buffer with 0.5 mL of apple juice
(3) describe the Michaelis−Menten mechanism for enzyme extract (the enzyme)
catalysis, including the meaning of the Michaelis− During this phase, the instructor may choose to give the
Menten parameters students the opportunity to make some experimental decisions.
(4) determine which apple has the slowest rate of browning, For example, the students can make the catechol solution and
making it the best choice for a fruit salad design and make the buffer solution, or the instructor can
(5) determine the mechanism of inhibition for a particular prepare the buffer in advance to save time. The students can
inhibitor based on changes in Michaelis−Menten decide how to best extract the catecholase from the apple (i.e.,
parameters juice the apple) or be provided with a protocol. If the
experiment is carried out with the whole class simultaneously,
Description of the Experiment the students can decide how to divide up the necessary tasks,
The experiment discussed in this paper is rooted in these two such as solution preparation or which students will test which
questions: mixtures.
After collecting the kinetic data, the Thinking About the
• Which apple would be best for a fruit salad?
Data questions lead the students to describe the effect of the
• What is the best way to keep cut apples from browning? enzyme on the oxidation reaction and to predict how to inhibit
The experiment has four cycles, each of which follows the the enzymatic catalysis of the oxidation. Students are asked to
format in Figure 1. Each cycle focuses on the kinetics of the decide whether or not to repeat the experiment using buffered
oxidation of catechol to quinone under different reaction or unbuffered mixtures based on their results from Cycle One;
conditions. The heart of the experimental protocol is the same the question foreshadows enzyme inhibition (Cycles Three
for each cycle: Students monitor the time-dependent UV−vis and Four). Data modeling takes place in Cycle Two, described
absorbance at 540 nm of mixtures of catechol with catecholase next. Thus, instructors could implement Cycle One as a
extracted from apples. Any spectrometer may be used; a standalone inquiry experiment for an entry level chemistry
spectrometer with a kinetics package is preferable because the laboratory course.
data are recorded in tabular form. The quinone produced in Cycle Two
the oxidation of catechol is colored, so the data collected
Data modeling using the Michaelis−Menten reaction scheme
represent the rate of change in the concentration of quinone.
is introduced in Cycle Two. In answering the Pre-Experiment
Students are required to make a prediction of an experimental
questions, students predict the appearance of a graph of the
outcome in each cycle. They also have opportunities to make
initial reaction rate versus substrate concentration after
decisions about how to carry out the experiment. For each
examining the Michaelis−Menten equation, which is provided
cycle, we summarize the pre-experiment questions, the
to students without derivation. The derivation of this equation
predictions made by the students, the variations of the basic
from the mechanism is done by students in the Post-
protocol, and the “Thinking About The Data” questions; the
Experiment questions.
steps in each cycle are made bold.
To test their prediction, the students repeat the protocol
Detailed options for timing for this experiment are described
from Cycle One but with varying amounts of substrate.
in the Supporting Information (instructor handbook).
Students may also be given the option of choosing which apple
Completing all four cycles is expected to require three to
varietals to test. The students decide which substrate
four 3 h lab periods. If solutions are prepared for students, the
concentrations to test given a range of concentrations;
first two cycles can be completed through the second round of
instructors should encourage students to be sure to obtain
data collection in one 3 h lab period. The modeling for Cycle
initial rates for enough trials at low substrate concentrations.
Two and prelab experiment questions for design Cycles Three
As noted in Cycle One, students can choose to run the
and Four could then be completed out of class or in a second
experiment in unbuffered mixtures, but if the apple varietals are
lab period. Alternatively, a single 4 h lab period is sufficient to
acidic, there may be little to no browning observed.
carry out the first two cycles, including the modeling. Data
The Thinking About the Data questions in Cycle Two
collection for Cycles Three and Four can be completed in a
guide students through linear and nonlinear data analysis to
single 3 h lab period, but students must be well-prepared and
arrive at the Michaelis−Menten kinetic parameters, KM and
efficient in order to obtain quality data.
vmax. Students are guided to linearize the Michaelis−Menten
Cycle One equation and construct a double-reciprocal plot of 1/vinit vs 1/
The experiment handout begins with a brief introduction to [S]. This Lineweaver−Burk plot is used to obtain a reasonable
the chemical basis of the browning of fruit. Students then estimate of the KM and vmax. An example of a Lineweaver−Burk
answer Pre-Experiment questions designed to get them plot based on pH ∼ 7 data from multiple student groups is
thinking about the oxidation of catechol to produce quinone displayed Figure 2. All student groups used the same buffered
and about enzyme kinetics. They are prompted to suggest a apple solution.
method for monitoring the reaction progress over time, and Students use the resulting KM and vmax estimates to fit the
they review the protocol. The students are required to predict full (nonlinear) Michaelis−Menten equation to the data,
the appearance of the absorbance versus time graph for a refining their parameters to get the best fit curve. The
solution of catechol and enzyme. To test this prediction, nonlinear analysis requires a minimization routine such as
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Cycles Three and Four

The experiment is extended in these two cycles by including
enzyme inhibition. The two cycles are motivated by reminding
students that all apples brown over time, so cooks may try to
find ways to slow the browning process. Pre-Experiment
questions guide students to select an inhibitor, recalling the
effect of acidity on browning from the first two cycles. Next,
students must decide (predict) the appropriate substrate and
inhibitor concentrations before going on to determine the
Michaelis−Menten kinetic parameters experimentally. The
inhibitor concentration must be selected so that the browning
reaction is slowed but not stopped. Students repeat the same
protocol to find the best inhibitor concentration and analyze
the results by answering the next set of Thinking About the
Figure 2. Typical Lineweaver−Burk plot based on student data Data questions. Students have more difficulty determining the
(uninhibited). Results from four groups were combined to give KM best concentrations than with any other aspects of this
and vmax of 1.37 × 10−3 M and 6.03 × 10−3 M s−1, respectively. These experiment; they often say something like, “just give me the
parameters were extracted via a simple linear regression (i.e., using the instructions.” Although it would be straightforward to simply
Excel LINEST function). provide appropriate concentrations, the point is to facilitate
students’ development of the skills that are more authentic to
Microsoft Excel’s solver, and the Instructor’s handbook research environments. The goal is to communicate to
includes a sample spreadsheet. Figure 3 shows the data from students that learning the process of experimental design is
important, not just getting the “right” answer.
Inhibition mechanisms are introduced in the Cycle Four
Pre-Experiment questions, which come after students select
their inhibitor and determine its appropriate concentration. A
series of questions guide students to match the inhibition
reaction mechanisms to the mechanism type (competitive,
uncompetitive, or noncompetitive) and the corresponding
equilibrium expression. Ultimately, students predict how their
initial rate versus substrate concentration graphs will be
affected by each of the inhibition mechanisms. At this point,
they set up the appropriate concentrations of enzyme,
inhibitor, and substrate in order to determine the mechanism
for their selected inhibitor. The students repeat the protocol
from Cycle Two to determine the Michaelis−Menten kinetic
parameters in the presence of the inhibitor; the final Thinking
About the Data questions guide the students to determine the
Figure 3. Typical Michaelis−Menten plot resulting from student data inhibition model from their results. The selection of inhibitors
(uninhibited) for apple solutions buffered at pH = 7. The data from has been guided by those commonly seen in commercial food
four student groups were combined. The extracted KM and vmax via products or recipes (citric acid, ascorbic acid, sodium benzoate,
the nonlinear regression were 1.97 × 10−3 M and 7.36 × 10−3 M s−1.
and lemon juice). Citric acid and lemon juice show
competitive inhibition, while ascorbic acid and sodium
benzoate show noncompetitive inhibition. Student data are
not always of sufficiently high quality to determine the mode of
Figure 2 plotted as vinit versus [S], including the nonlinear
inhibition, but students are able to reflect on why their
fitted curve. Using the M−M equation rather than its linearized
experimental design was insufficient and what changes they
cousin results in a fit that does not overemphasize low
could make to achieve better outcomes.
substrate concentrations (large 1/[S], where the initial rates Students observe inhibition as shown in Figure 4a; Figure 4b
have a larger relative error compared to high substrate demonstrates that these students were able to determine that
concentrations). citric acid inhibited browning through a competitive
The data presented in Figures 2 and 3 were collected using mechanism. However, some students do find that their
the same apple extract due to the simultaneous (as opposed to collected data make it challenging to clearly distinguish
rotational) structure of the laboratory course. Thus, it is between mechanisms. One important reason for this is that
possible for different teams of students to directly compare the system studied is an “apple”, not a well-controlled system.
their data for validity. Another issue is that students frequently neglect to collect
This cycle closes with an open-ended question, “Suppose uninhibited data when collecting the inhibited data and
you want to make a fruit salad with apples. What factors would therefore compare their inhibitor results to prior results with
you consider when preparing the apples and the salad? Discuss a different apple, albeit the same type of apple. This resultor
based on your kinetics results.” Thus, instructors in a physical rather, that lack of a clear resulthelps students learn to justify
chemistry laboratory course could choose to only carry out their conclusions as well as recognize some of the challenges in
Cycles One and Two, which would answer the question of the experimental design and inherent to working with natural
experiment title and include data modeling. samples.
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the inhibitor cycles is a good option for simultaneous mini-

research projects; different student teams may select the
inhibitor of their choice and present their results to the whole
class.
Students are prompted to share results in several instances in
the experiment handout, as is typical for POGIL−PCL
experiments.25 However, we intentionally wrote this experi-
ment so that it could also be implemented in courses using
experiment rotation. To facilitate comparisons of parameters
from different apples, instructors required students to share
data in a common spreadsheet that was updated by students
over the course of the semester or by posting results on a
discussion board using the course management system. In this
way, each team of students obtains quality data to analyze, and
Figure 4. Typical Lineweaver−Burk plot based on student data the data set builds as new teams rotate through completing the
(uninhibited and inhibited with citric acid). The main graph is experiment. Scheduling was done so that all teams, including
enlarged to focus on data within a similar substrate concentration the first team, had access to data from a different apple varietal
range; the inset shows the full data set. to compare parameters for different species in order to

■ HAZARDS AND SAFETY PRECAUTIONS

Students should be aware that catechol (MSDS 04360) and
complete the required analysis, which was included in their
laboratory reports. The instructor also provided oversight to
ensure different apple varieties and different inhibitors were
benzoquinone (MSDS 96461) are acute eye, skin, lung, and selected by students at different times. Although the
gastrointestinal irritants. In addition, benzoquinone is an opportunity for whole class discussion was lost, completion
environmental toxin. Appropriate PPE should be worn by all and submission of the prelaboratory questions in advance of
individuals in the laboratory, including goggles and nitrile the laboratory provided TAs the opportunity to respond to
gloves, to limit exposure to catechol and quinone during the student ideas and ask further questions as needed.
experiment. Waste should be disposed of properly to avoid
releasing benzoquinone into environmental water.
Although apple contents are benign, students should be
■ ASSESSMENT OF LEARNING OUTCOMES
The primary mode of evaluation of student achievement of
reminded that they are not to eat, drink, or ingest any material outcomes was through analysis of submitted lab reports using a
in the laboratory environment irrespective of its role as a food detailed grading rubric (two examples are included in the
product in a context outside the lab.

■
Supporting Information). The initial rubric was based on an
Excel rubric published in this Journal.27 The published rubric
IMPLEMENTATION was modified using categories from the ELIPSS rubrics28,29 to
Different variations of the experiment were implemented at add criteria to reflect explicitly the focus on critical thinking
multiple institutions over the last 15 years. The POGIL−PCL and argumentation. The grading rubric has also been imported
version was used at over five institutions with over 400 into the learning management system to facilitate scoring and
students. It was implemented multiple times both with all feedback to students. Instructors’ evaluation of student reports,
students in a section completing the experiment simulta- in both course settings, showed that students were generally
neously and in a rotation style where different student teams successful with respect to achieving the desired process and
complete the experiment in different weeks throughout the content skills.
semester. In addition, this experiment was tested with about 50 Students successfully analyzed and manipulated equations
faculty at three different POGIL−PCL workshops. and graphical representations to model experimental results,
This experiment can be implemented in full, which takes including using the parameters extracted from the Line-
three or four laboratory sessions (each typically about 3 h in weaver−Burk plot to inform their initial guesses for the
length) depending on how much time students are given in the nonlinear fit of the data to the Michaelis−Menten equation.
laboratory to work on data analysis. The first cycle could be Students were able to successfully choose experimental
used in an introductory laboratory course as long as the conditions to obtain appropriate data for analysis most of
instructor provides the buffer solution. The first and second the time, although they often struggled to articulate the
experiment cycles could be implemented as a standalone rationale for their choices. It was also observed that students
experiment in one or two lab-class periods as part of a standard tend to want to use serial dilutions, even when cued to
physical chemistry laboratory sequence. The instructor hand- consider that this approach will not generate the most useful
book, included in the Supporting Information, provides data set.
suggestions based on different institutional constraints. One area of concern, based on student reports, was the
Although not always possible, carrying out the experiment tendency of students to neglect explicitly addressing the
with the whole class simultaneously has some advantages. The guiding question of the activity. The students’ focus on
instructor leads a discussion of the pre-experiment questions equations is problematic given that a fundamental practice in
and guides students as they brainstorm experimental choices, science is to ask questions to inform investigations. The
especially making the buffer and arriving at a consensus National Academy of Sciences recommends that students build
decision regarding buffering in the second cycle. Doing the competence in asking questions to inform investigation, listing
experiment with the whole class simultaneously makes it easier this competence as one of eight science and engineering
for students to study different apples of their choosing. essential practices.23 Thus, instructors should tailor their
Students can be prompted to share their predictions. Including assessment tools (i.e., rubrics) to guide students to explicitly
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use their results to address the guiding questions. This Notes

experiment and associated assessment of student work may be The authors declare no competing financial interest.

■
considered part of the broader effort to lead students away
from viewing the purpose of a laboratory activity to “get a
ACKNOWLEDGMENTS
number” or a set of numbers and toward viewing it in a way
that is conducive to using, and interpreting, evidence to We acknowledge financial support from National Science
address research question(s). Foundation grants DUE-1044624 and DUE-

■
1726066,1726071. We appreciate the efforts of the many
CONCLUSIONS students and instructors who have completed the experiment
and provided valuable feedback over the years.

■
The experiment we describe here is guided inquiry (with some
elements of open inquiry); can be completed in two to four
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