Laboratory Exercise
Todd P. Silverstein*
Myoglobin Structure and Function: A
Multiweek Biochemistry Laboratory
Projectw
s
From the †Department of Chemistry, Willamette University, Salem,
Oregon, 97301, From the
Abstract
We have developed a multiweek laboratory project in
which students isolate myoglobin and characterize its
structure, function, and redox state. The important labora-
tory techniques covered in this project include size-
exclusion chromatography, electrophoresis, spectropho-
tometric titration, and FTIR spectroscopy. Regarding pro-
tein structure, students work with computer modeling
and visualization of myoglobin and its homologues, after
which they spectroscopically characterize its thermal
denaturation. Students also study protein function (ligand
Keywords: inquiry-based teaching; biophysical methods; protein
structure function and folding; computational biology; active
learning; laboratory exercises
Introduction
The relationship between protein structure and function
has been a crucial aspect of biochemistry for the last 50
years or more. In the modern undergraduate biochemistry
course, discussion of the isolation, characterization, struc-
ture, and function of proteins takes up over one-fourth of
the typical one-semester course. Laboratory projects focus-
ing on computer modeling of protein structure have been
increasingly common in the literature since about 2000
[1–7]. Almost all biochemistry laboratory texts feature proj-
ects in which students isolate a protein from a natural
source, purify it, and then assay its biochemical activity
[8–15]. In addition to these independent projects that last
223 laboratory periods and are centered on individual
w
s Additional Supporting Information may be found in the online
version of this article.
Scott C. Meyer’s current address is: Department of Chemistry,
Benedictine University, Lisle, IL, 60532
*Address for correspondence to: Department of Chemistry, Willamette
University, Salem, OR 97301. E-mail: tsilvers@willamette.edu
Received 25 July 2014; Accepted 2 November 2014
DOI 10.1002/bmb.20845
Published online 27 February 2015 in Wiley Online Library
(wileyonlinelibrary.com)
Biochemistry and Molecular Biology Education
Sarah R. Kirk
Scott C. Meyer
Karen L. McFarlane
Holman
binding equilibrium) and are instructed on topics in data
analysis (calibration curves, nonlinear vs. linear regres-
sion). This upper division biochemistry laboratory project
is a challenging and rewarding one that not only exposes
students to a wide variety of important biochemical labo-
ratory techniques but also ties those techniques together
to work with a single readily available and easily charac-
terized protein, myoglobin. V C 2015 by The International
Union of Biochemistry and Molecular Biology, 43(3):181–
188, 2015.
enzymes, several semester-long biochemistry laboratories
devoted to a single protein or enzyme have been published
[8, 16–25]. Although the thematic unity of working on one
specific protein for the whole semester is attractive, not all
important experimental methods can be applied to a single
protein.
Therefore, instead of designing an entire course around
one protein, we decided to embed an extended protein
structure/function project within our semester-long upper
division laboratory course, Experimental Biochemistry I
(CHEM 346). We devote six 3-h laboratory periods in this
course to an extended project on myoglobin (Mb). The
remainder of the course includes a number of other multi-
lab period projects that introduce our students to experi-
mental methods such as HPLC, GCMS, phospholipid mem-
brane dynamics, laser light scattering, spectrophotometric
and fluorimetric determination of enzyme activity, and
electrochemical biosensors.
The heme protein myoglobin is found in most muscle
tissue. Like hemoglobin (Hb), Mb contains a heme-bound
Fe(II) cation that can be oxidized to the Fe(III) form
(metMb). For many decades it was believed that the main
function of the Mb-heme-Fe(II) cofactor was to bind O2, as
well as CO, N2, nitrite, and azide ligands. Recent evidence
suggests that in fact, the major biological function of Mb
may be to catalyze the nitrite/nitric oxide interconversion
181

in muscle [26, 27]. Unlike Hb, which is tetrameric and
binds oxygen cooperatively with a Hill coefficient of n 5 2.8,
Mb is monomeric and binds oxygen noncooperatively
(n 5 1). Mb is easily extracted from muscle tissue and spec-
trophotometric characterization of the Fe-heme is facile, as
it absorbs strongly in the visible and UV ranges. In fact, the
visible absorption of heme-coordinated Fe(II) in Mb is
responsible for the reddish appearance of fresh (reduced)
meat, whereas the visible absorption of heme-coordinated
Fe(III) in metMb is consistent with the brownish color of
old (oxidized) meat [28, 29].
We describe here our six-period project, in which stu-
dents isolate Mb from ground beef [29], spectrophotometri-
cally characterize its oxidized and reduced forms [29],
probe its ligand binding equilibrium [30], perform molecu-
lar modeling on its structure and that of its homologues
[31], and use FTIR to characterize its secondary structure
in the native and denatured forms [32]. We have based our
extended Mb project on the four papers cited above
[29–32], adapting and expanding the projects beyond what
is described in the original source papers. Specifically, we
have added new methods of analysis (e.g., SDS-PAGE),
adapted to changes in molecular modeling websites, and
added extensive quantitative data analysis and curve-fitting
components.
Student Learning Objectives:
1. Proficiency in carrying out the following biochemical
techniques: centrifugation and protein isolation; column
chromatography and SDS-PAGE (proteins); and use of
protein structure databases
2. Strengthened understanding of the following biochemical
topics: protein structure modeling, characterization, and
denaturation; protein homology and evolution; and
protein-ligand binding equilibrium
3. Increased competence in biochemical data analysis,
including: x-y scatter plots and curve fitting (both linear
and nonlinear regression); and calibration curves
4. Increased confidence and mastery in using the following
instruments: UV-Vis and FTIR spectrophotometers; gel
electrophoresis; and in silico computer modeling of pro-
tein structure
5. Enhanced ability to: employ critical thinking and scien-
tific reasoning; speak and write effectively about scien-
tific concepts.
Teaching and Laboratory Procedures
This extended laboratory project centered around Mb
requires six 3-h laboratory periods, which include some in-
class time for data analysis. Detailed instructions are pro-
vided in the Supporting Information. In laboratory Period 1,
students extract Mb from ground beef, reduce, and oxidize
portions of the sample, use a Sephadex column to “desalt”
the excess oxidizing agent, and use UV-Vis absorbance to
182
Biochemistry and
Molecular Biology Education
characterize the reduced and oxidized samples. In Period 2
students employ SDS-PAGE to characterize the extracted
samples. In Period 3, azide binding to metMb is examined
via spectrophotometric titration. In Periods 4 and 5, stu-
dents use online databases, websites, and software to char-
acterize the structure of Mb and a selected novel homolog.
In the final period, students employ FTIR spectroscopy to
probe protein secondary structure in both the native and
thermally denatured conformations.
Students work in pairs throughout this experiment.
Because use of the FTIR spectrophotometer takes up all
3 h of Period 6, we can only schedule use of the instru-
ment if students are paired. During Periods 1 and 3, each
student pair has access to its own small Thermo-Electron
Genesys-10 UV-Vis spectrophotometer. In Period 2, all
pairs mount their gels and run from a single electrophore-
sis power supply. In Periods 4 and 5, paired students sit
adjacent to each other, but each works on his/her own
desktop computer in our computer room. We have run
this project six times, every spring since 2009. Course
enrollment has varied from 7 to 20 students, averaging
10212.
Period 1, Part IA: Myoglobin Extraction,
Separation, and Spectral Characterization
To begin the project, students extract Mb from ground beef
in a single 3-h laboratory period, following a modification
of the protocol of Bylkas and Andersson [29]. In this part of
the project, students work from the original literature
source; their laboratory manual contains screenshots of
selected paragraphs taken directly from the Bylkas and
Andersson paper (see Supporting Information). A portion of
the extracted Mb is fully reduced from Fe(III) to Fe(II) with
dithionite (turning deep red) and a separate portion is fully
oxidized to Fe(III) with ferricyanide (turning brown). Fol-
lowing oxidation, the metMb is separated from excess ferri-
cyanide using a Sephadex G-25 size-exclusion gel chroma-
tography “desalting” column. The three forms of Mb
(original extract, reduced, and oxidized) can then be char-
acterized spectrophotometrically. Reduced Mb features O2
bound to the Fe(II) center in the heme. It has prominent
peaks in the visible range at about 540 and 580 nm, and its
Soret band is at 417 nm with e417 5 128,000 M21 cm21
[29]. The oxidized metMb has H2O bound to the Fe(III)-
heme; its visible peaks appear at 505 and 620 nm, and its
Soret band is at 409 nm with e409 5 179,000 M21 cm21
[29]. Given the prominent changes in the wavelength and
molar absorptivities of the Soret band absorbance peaks, it
is straightforward to spectrophotometrically determine the
concentrations of Mb and metMb, and monitor changes in
Fe(II)/(III) redox chemistry and ligand binding.
Period 2: SDS-PAGE
In a subsequent 3-h laboratory period students use SDS-
PAGE to separate and visualize the protein components of
both the original Mb extract, and the desalted metMb
Myoglobin Structure and Function
 SDS-PAGE gel of Mb solutions (representative stu-
FIG 1 dent results). Lanes 1 and 8: Novex protein stand-
ards; Lanes 2 and 9: PP1 protein standards; the
three smallest standards are 10, 15, and 20 kDa;
Lane 3: Mb (horse skeletal muscle, Sigma M-0630);
Lane 4: supernatant of ground beef extraction;
Lane 5: supernatant 1 dithionite; Lanes 6, 7, and
10: 10, 20, and 15 mL (respectively) of superna-
tant 1 ferricyanide, Sephadex G25-desalted. In
Lanes 327 and 10, MW(Mb) 5 16.2 6 1.8 kDa; in
Lanes 427 and 10, the Mb band accounts for
20 6 3% of the protein in each lane.

column eluent. Here students are introduced to electropho-

resis, including loading and running the gels, quantifying
TM
the bands (using GelDoc ), and calculating molecular
weights and protein composition of each mixture (Fig. 1).
Students determine the molecular weight of Mb in their
mixtures and compare it to the literature value (17.6 kDa
for Sigma M-0630); they also determine the percentage of
Mb in each mixture (20 6 3%), showing that the desalting
Sephadex G-25 column merely serves to remove the excess
ferricyanide, but does not appreciably separate any of the
proteins in the mixtures.
Period 3, Part IB: Myoglobin Function: Ligand
Binding
Students perform a spectrophotometric titration to monitor
the binding of azide to Fe(III) in metMb and characterize
the azide/metMb binding equilibrium. This modification of
the experiment described by Marcoline and Elgren [30]
also takes a single 3-h laboratory period. By following
changes in the 540 (or 580) nm peak, students can calcu-
late r, the ratio of bound ligand to total protein, and [L]free,
the equilibrium concentration of free (unbound) azide
ligand. Fitting the r vs. [L]free plot to the equation for hyper-
bolic saturation [Eq. (1)] allows a best fit determination of
the protein-ligand dissociation equilibrium constant, Kd,
and the number of bound ligand molecules per protein, n.
n n½Lfree
r5 5 (1)
11Kd =½Lfree ½Lfree 1Kd
Typically, a linearized form of the results, such as a
Scatchard plot or a double-reciprocal plot, would be used
to determine n and Kd. However, it has been pointed out
MetMb/azide spectrophotometric titration (repre-
FIG 2 sentative student results). Total initial [Mb] 5 16.1
mM; changes in metMb absorbance at 543 nm
were followed upon addition of azide aliquots
amounting to final [N3-] 5 2 – 948 mM. (a) r vs.
[N3-]free fit to the equation for hyperbolic saturation
gives Kd 5 35.6 6 2.0 mM, n 5 0.973 6 0.013, and
R2 5 0.997. (b) The double-reciprocal plot, 1/r vs. 1/
[N32]free gives intercept 5 1.9 6 0.8, slope 5 21 6 3
mM21, and R2 5 0.83 for all 12 points (solid line);
this yields calculated values of n 5 0.52 6 0.22 and
Kd 5 11 6 5 mM. Omitting the two lowest concentra-
tion points (1.1 and 3.9 mM free azide) gives inter-
cept 5 1.075 6 0.023, slope 5 32.4 6 0.5 mM21, and
R2 5 0.998 (dashed line); this gives calculated val-
ues of n 5 0.930 6 0.020 and Kd 5 30.1 6 0.8 mM. A
Scatchard plot of these data (r/[N32]free vs. r) gives
similar large error in the two lowest concentration
points. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
many times that linearizing data often magnifies error in
the derived parameters [33–35]. Students are therefore
instructed to analyze their results using both linear regres-
sion of linearized data and nonlinear regression of the
hyperbolic curve, and discuss which is more reliable. Stu-
dents find that n does indeed equal one for azide binding to
metMb, and Kd is in the 10–100 mM range (see Fig. 2), in
agreement with the literature (4–80 mM) [36]. Figure 2b
clearly shows how error is magnified in the double-
reciprocal plot for the two lowest concentration points.
Even omitting these points gives errors, relative to the

Silverstein et al 183
 Biochemistry and
Molecular Biology Education

Mb structure comparison. (A) solved structure of sperm whale Mb, pdb file 1VXA; (B) Swiss model predicted structure
FIG 3 of Mb from unicorn icefish (Channichthys rhinoceratus), UniProt file E5G628. Color key for amino acid side chain types
at the protein surface: white 5 hydrophobic; red 5 negative; blue5 positive; pink 5 polar neutral; yellow 5 aromatic;
green 5 proline. Note the heme binding cavity in the center of both structures; most of the surface directly above this
cavity is identical in both (A) and (B). Besides these and other similarities, six key differences between the two forms of
Mb are marked above.

values derived from nonlinear regression, of 215% and
24.5% in Kd and n, respectively; the Scatchard plot is a lit-
tle better, with errors of 29% and 21.7%, respectively.
Part II: Myoglobin Three-Dimensional Structure:
Native vs. Denatured
Myoglobin Structure on the Web
In 1998, Leon et al. published a biochemistry laboratory
project in which students used internet websites and free-
ware to model protein structure [31].1 Leon et al. describe
a project in which students select a protein whose structure
has already been solved and deposited in the Protein Data
Bank (PDB). They then carry out a series of four operations
on the internet: (a) use search programs to find a “novel
homolog,” i.e., a homologous protein whose structure has
NOT yet been solved; (b) submit the amino acid sequence
of the novel homolog to secondary structure prediction pro-
grams and compare secondary structures of the original
protein and its novel homolog; (c) submit the amino acid
sequence of the novel homolog to Swiss Model to obtain a
predicted three-dimensional structure; and finally (d) com-
1 [2, 37238]. This program not only allows homologous pro-
In order to increase the diversity of protein structures exam-
ined, we have half of the students start with myoglobin, a pre-
dominantly alpha-helical protein, and the other half start with
the predominantly beta-sheet protein, lysozyme.
pare the 3D structure of original protein to that of its novel
homolog.
Periods 4 and 5
It takes students about 4.5 h to finish operations (a–d) listed
above. We have our students use the final 1.5 h in laboratory
period 5 to prepare figures for an oral presentation in which
they carefully characterize the major differences and simi-
larities between the original protein and its novel homolog.
All of the websites described by Leon et al. have
changed dramatically in the nearly 2 decades since the
original publication. Some things have gotten easier. For
example, in 1998 one had to visit three separate websites
to obtain a protein’s structure coordinates (PDB), structure,
and function information (UniProt/SwissProt), and perform
homology searching (Blitz). Currently all of this information
can be obtained from the PDB website and links therein.
On the other hand, other aspects of the project have gotten
more difficult. The protein visualization program recom-
mended by Leon et al., Rasmac, is woefully outdated; in
addition, a useful protein structure comparison program
that we discovered around 2000, Protein Explorer, has not
been supported since about 2006. However, an even better
freeware program, PyMOL, has since come into wide use
teins to be aligned and visualized, but it also has many dif-
ferent viewing modes that can be implemented. We encour-
age students to explore the capabilities of PyMOL on their

184 Myoglobin Structure and Function

own, but we require that they use at least three different Hazards
views to compare their two proteins: van der Waals sur-
face; electrostatic surface; and polarity/hydrophobicity (Fig.
Compound Danger Hazard rating
3). The former two views are built into PyMOL, and the lat-
ter is provided by the “color_by_restype” script written by Sodium Eye damage; skin NPFA hazard 5
researchers at Queens University in Kingston, Ontario [39]. dithionite irritation Harmful 2, GHS eye
(bisulfite) if swallowed, inhaled, damage 5 1
Structure of Native and Unfolded Myoglobin or absorbed through skin
Characterized by FTIR Sodium azide Fatal if swallowed, or GHS dermal
In the sixth and final 3-h laboratory period of our Mb absorbed through skin toxicity 5 1,
structure and function project, students use FTIR to GHS oral
observe spectroscopic differences in native, folded pro- toxicity 5 2,
GHS aquatic
teins that are predominantly alpha-helical (e.g., Mb) and
toxicity 5 1
those that are predominantly beta-sheet (e.g., lysozyme). Potassium Harmful to aquatic life GHS aquatic
Students can determine the actual percent helix vs. sheet ferricyanide toxicity 5 3
by accessing structural information at the Protein Data
Bank (pdb) website, and compare this to the FTIR spectro-
scopic results that they get for each protein. They then
incubate these proteins at elevated temperatures and use Common Pitfalls
changes in FTIR peaks to characterize the thermal dena-
turation process (Fig. 4a).
Project
This final sub-project is based on the experiment
Section Problem Solution
described by Olchowitz et al. [32]. We have made two major
modifications in order to dramatically improve data analysis. Part IB: At lowest azide Set these free azide
First, we found that in order to get the expected sigmoidal concentrations, concentrations
changes in FTIR peaks with changes in temperature, careful calculated to the minimum
baseline subtraction, and normalization is required. In Fig. concentration value, zero.
of free azide is
4a the spectra are baselined at 1700 cm21 and normalized
negative.
at the a-helix amide I peak, 1651 cm21. We describe these Part IIA Amino acid Find and omit
processes in detail in our Supporting Information. Second, sequence the N-terminal
we encourage our students to analyze their results in order differences signal sequence.
to obtain a denaturation temperature, Tden (also called TU or between UniProt
Tmelt in the literature); this is the temperature at which the vs. PDB sites.
equilibrium between native/folded protein and denatured/ Swiss Model fails Select a different
unfolded protein features a 50/50 mixture. template
The denaturation equilibrium, in which the folded, Part IIB Protein aggregates For T  85 C, incubate
native protein (N) denatures, and unfolds (U) can be char- at high T for only a few minutes.
Determining the Have students consider as
acterized by an equilibrium constant (Kden 5 KU 5 [U]eq/
effect of T on many spectral changes
[N]eq, and by an enthalpy and entropy for the process, DH U protein structure: as seems productive. We
and DS U. Given a change in some FTIR spectroscopic plotted the relative
parameter y as the protein unfolds, the dependence of the absorbances of
denaturation equilibrium on T, TU, and DH U, is shown in three different peaks
Eq. (2) (see Supporting Information, Appendix 4): in Fig. 4b, but
DH 
the shift in kmax of
U 1 1
R ðTU 2T Þ the amide I peak
yloT 1yhiT  e
y5 DH 
(2) can also be
U 1 1
R ðT U 2 T Þ
11e informative [32].
Using nonlinear regression to fit the data in Fig. 4 gives
best fit values for both TU and DH U. Students can then
compare their results to literature values for Mb unfolding Student Reports
in aqueous buffer: TU 5 81–83 C, DH U 5 100–130 kcal/mol For Part I (Mb extraction/characterization and azide bind-
[40242]. Student results suggest (Fig. 4b) that in D2O, Mb ing), students write informal lab reports in which they
unfolds at a lower temperature (65–70 C) than it does in address questions focused on data analysis to hone their
aqueous buffer, but with a DH U that is the same or higher critical thinking skills. They use Beer’s Law to calculate Mb
(100–300 kcal/mol). concentrations, and discuss how heme-iron oxidation alters

Silverstein et al 185
 Biochemistry and
Molecular Biology Education

FTIR characterization of Mb thermal denaturation (representative student results). (a) Relative absorbance (Ak/A1651) of
FIG 4 60 mg/mL Mb in D2O, at eleven different temperatures, 22–87 C. Note the appearance, above 64 C, of shoulders at 1618
and 1680 cm21, and an aggregated protein peak at 1570 cm21. (b) The three wavelengths in the spectra in (A) that
change dramatically with T are plotted: R shoulder, A1618/A1651 (black circles); L shoulder, A1680/A1651 (blue diamonds);
and aggregated protein peak, A1570/A1651 (blue diamonds). Fitting the R shoulder points using Eq. (2) gives
DH U 5 290 6 70 kcal/mol and TU 5 65.79 6 0.15 C; fitting the L shoulder and aggregated protein points gives
DH U 5 120 6 50 kcal/mol and TU 5 68.3 6 1.2 C. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]

UV-Vis absorbance. Students use SDS-PAGE standard pro- Student Feedback/Assessment

tein mobilities to prepare a calibration curve to determine
molecular weights; they compare their Mb molecular
weight to the literature value, and also establish the frac-
tion of Mb in their samples. For Part IB, students use both
linear and nonlinear regression to determine Kd and n for
azide binding to metMb.
For Part II (protein structure modeling and thermal
denaturation) students prepare an oral presentation in
which they carefully characterize the major differences and
similarities between Mb and their chosen novel homolog.
They use FTIR amide I peaks to distinguish between pro-
teins that are primarily a-helix versus b-sheet, and to fol-
low temperature-driven changes in secondary structure.
Finally, students use nonlinear regression of their FTIR
peak versus T data to determine DH U and TU and compare
to literature values.
We assessed this project (and the entire Experimental Bio-
chemistry course sequence) in two ways: with a pre/post
laboratory skills exam, and with a survey of student
alumni. Of the 33 questions on the exam, 8 covered con-
cepts related specifically to this protein structure labora-
tory project (e.g., protein separation, structure characteri-
zation, ligand binding). On these eight questions, student
performance improved from 53% correct before the start of
the course, to 85% correct at the end (and 91% after the
second semester of the course sequence). In addition to
these exam results, as instructors of the course, we
observed consistently an increase in both skills and confi-
dence level as students moved through the course.
We carried out a survey of our alumni to assess the
impact of this laboratory sequence on their perceived knowl-
edge of techniques, biochemical topics, and instrumentation.

186 Myoglobin Structure and Function

We had 18 people respond out of a total 23 accessible Bio-
chemistry Track alumni over 4 years. From the survey
results, it was clear that this laboratory was successful in
meeting our intended goals. Students felt confident in their
understanding of all the major biochemical topics covered, as
well as in their ability to analyze biochemical data. One stu-
dent wrote the following about what s/he got out of the course:
“I have a solid foundation for lab technique and good lab
practices upon which to build, and have an ability to learn
new protocols quickly to become both proficient and efficient
with them.” Interestingly, while students rated highly their
knowledge of all of the instruments that they employed in the
course, they expressed the greatest confidence in their ability
to use the instruments that they were repeatedly exposed to.
In particular they rated the course highly for helping them to
develop critical thinking skills and scientific reasoning (4.6 on
a Likert scale of 1 to 5) and to speak about scientific concepts
(4.5). Students rated satisfaction with their ability to write sci-
entifically even more highly (4.8). In short, the results of our
pre/post laboratory skills exam and our alumni survey allow
us to conclude that this project did indeed achieve the learn-
ing objectives that we aimed for (see list above).
Further Extensions
We decided to focus our protein structure/function project on
myoglobin because it is readily obtained (from ground beef
and in purified form), easily characterized (by spectrophotom-
etry), and has been used in a number of published biochemis-
try laboratory projects. There are undoubtedly many other
proteins that fit this bill and could be substituted for Mb.
We have on occasion included another interesting labo-
ratory project dealing with protein denaturation. As pub-
lished [43], this project uses various concentrations of guani-
dine hydrochloride to denature chymotrypsin. Fluorescence
spectroscopy is then used to probe both the structure of the
protein as well as its enzymatic activity; the unique aspect of
this particular laboratory project is that students simultane-
ously assay changes in both protein structure and function
as a result of denaturation. Although we have not adapted
this technique to Mb, we believe that it would not be diffi-
cult. In fact, due to the intrinsic absorbance of the Fe(II/III)
center in Mb, UV-Vis spectrophotometry could probably be
used in place of fluorimetry. In order to probe the effect of
denaturation on protein structure, one would simply follow
changes in the UV-Vis (or fluorescence) spectrum with gua-
nidine concentration. To probe the effect on protein func-
tion, one would select a few representative guanidine con-
centrations and perform azide binding titrations at each
one. Presumably, as denaturation increases, the Kd for azide
binding should also increase (weaker binding).
Summary
We have developed an extended project for an upper divi-
sion biochemistry laboratory course that uses Mb as a cen-
Silverstein et al
tral theme to investigate several important experimental
techniques, as well as the relationship between the struc-
ture and function of a protein. Over six 3-h laboratory peri-
ods, the students isolate Mb from muscle tissue, spectro-
scopically characterize the oxidation state of its heme
moiety and its ligand binding, analyze the protein’s struc-
ture via molecular modeling programs, and track its ther-
mal denaturation with FTIR. We have expanded and com-
bined previously described experiments [29232], and also
updated procedures to reflect changes in the availability of
structure analysis software like PyMOL [2, 37, 38]. This
project has the advantage of fulfilling two sometimes dis-
parate goals in laboratory curriculum development: It
introduces students to a wide variety of biochemical techni-
ques, while at the same time organizing the project around
a central, biologically relevant theme.
Acknowledgements
The authors thank Willamette University for its extensive
long-term support of laboratory curriculum development,
and our colleague Dr. Alison Fisher for her help with this
manuscript. This material is based upon work supported
by the National Science Foundation under Grant No. DUE-
1044737. Any opinions, findings, and conclusions or recom-
mendations expressed in this material are those of the
authors and do not necessarily reflect the views of the
National Science Foundation.
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