426 Journal of Chemical Education Vol. 74 No.

4 April 1997
In the Laboratory
Microburger Biochemistry: Extraction and Spectral
Characterization of Myoglobin from Hamburger
Sheri A. Bylkas and Laura A. Andersson*
Department of Biochemistry, 103 Willard Hall, Kansas State University, Manhattan, KS 66506
In this experimental protocol, we present a biochemis-
try experiment that provides a simple demonstration of use-
ful biochemical methods. The procedure is designed to be
undertaken at one of two levels of complexity, depending
upon the instrumentation available for student use and the
intended course level. The basic experiment is designed
for an advanced secondary school laboratory or a beginning
college biochemistry

/

biology laboratory (perhaps a general
organicbiochemistry service course) that uses a single-
beam spectrophotometer such as a Spectronic 20.
1
The pro-
tocol combines protein extraction, a partial protein purifi-
cation procedure, an oxidation and reduction scheme, and
simple spectroscopic analysis. The advanced experiment
is designed for an intermediate or advanced college bio-
chemistry laboratory that permits hands-on student use of
a research-grade scanning spectrophotometer such as a
Shimadzu UV2101 or Hitachi U3200 (perhaps in teams of
two students). The latter experiment includes not only tech-
niques listed for the basic experiment, but also gel filtra-
tion chromatography and generation and analysis of spec-
tral scans.
Mammalian myoglobin (Mb) is a monomeric oxygen-bind-
ing protein found in cardiac and skeletal muscle. Its pri-
mary function is to store oxygen until it is required by the
tissue. The protein has a single heme group (iron protopor-
phyrin IX) that is ligated to the protein by histidine resi-
due His-93. The experiment illustrates the two common and
stable forms of the protein, Fe
3+
H
2
OMb (Met-Mb) and
Fe
2+
O
2
Mb (Oxy-Mb), shown in Figure 1. In vivo, Oxy-Mb
and deoxy-Mb (the unligated ferrous state) are the two most
common forms. However, in a nonliving system, Oxy-Mb is
slowly converted to Met-Mb as the heme-bound O
2
molecule
is released and an active-site H
2
O molecule is bound.
Myoglobin is easily extracted from a lean ground steak
patty.
2,3
The meat contains very high levels of both Oxy-Mb
and Met-Mb, which have unique spectral properties (see
Table 1) that can be used to accurately identify and charac-
terize the sample identity with respect to both oxidation
state and functional state.
One can use chemical agents to duplicate the oxidizing
and reducing reactions of living systems. The functionally
important state, Oxy-Mb, can be converted to inactive Met-
Mb by oxidation in a reaction where an electron from the
(ferrous) iron is donated to the oxidizing agent (eq 1). Alter-
natively, Met-Mb can be converted to Oxy-Mb by a reduc-
tion reaction in which the heme iron gains an electron from
the reducing agent (eq 2). These reactions are illustrated
below.



e

Fe
2+
-O
2
-Mb + ox. agent (K
6
[FeCN
6
]) Fe
3+
-H
2
O-Mb (1)

+

e

Fe
3+
-H
2
O-Mb + red. agent (Na
2
S
2
O
4
) Fe
2+
-O
2
-Mb (2)
After these reactions, the reducing and oxidizing
agents can be left in the sample for the basic lab. The stu-
dents in the basic lab will study only the visible region of
the Mb spectra, avoiding both the added spectral bands
from the (nonremoved) redox agents and the intense Soret
band (see Table 1). For the advanced lab, the redox reagents
are separated from the Mb sample by gel filtration chroma-
tography to yield homogeneous samples of the muddy
brown Met-Mb or bright red Oxy-Mb for detailed spectral
analysis, scanning the full range from 700 to 300 nm. The
advanced laboratory protocol also provides students with an
opportunity to use the BeerLambert law
A = cb
where A = observed absorbance; = extinction coefficient
(mol/Lcm)
1
; c = molar concentration; and b = path length
of cuvette in centimeters.
Because the color change between Oxy-Mb and Met-Mb
is so dramatic (illustrating the bright red natural color of
fresh meat vs. the aged brown color of older meat), it is
worthwhile for the instructor to demonstrate the gel filtra-
tion step. Method(s) used in this protocol are simple, rela-
tively inexpensive, and nonharmful to the student.
*Corresponding author. Current address: Department of
Chemistry, Vassar College, Poughkeepsie, NY 12601. Phone: 914/
437-5746; Email: LAAndersson@vaxsar.vassar.edu
a
Adapted from refs 1 and 2 and unpublished results (Andersson L.
A.; Bylkas, S. A.; Cole, A. B.).
b
Wavelength, in nm; extinction coefficient is in parentheses, in units
of mM
1
cm
1
.
s e x e l p m o C n i b o l g o y M r o f s k a e P n o i t p r o s b A . 1 e l b a T
a
e l p m a S n o i g e R e l b i s i V
b
n o i g e R t e r o S
b
b M - t e M 5 3 6 4 0 5 ) 9 7 1 ( 9 0 4
b M - y x O 0 8 5 2 4 5 ) 8 2 1 ( 7 1 4 8 4 3
(A) (B)
Figure 1. Representation of the heme environment for the two
states of myoglobin under investigation. (A) Met-myoglobin, [Fe
3+
-
H
2
O-Mb]; (B) Oxy-myoglobin [Fe
2+
-O
2
-Mb].
Vol. 74 No. 4 April 1997 Journal of Chemical Education 427
In the Laboratory
The Experiment
Teams of two students are suggested. A list of materi-
als needed for each student, assuming two-person teams, is
presented below.
Basic Laboratory
Myoglobin Extraction and Partial Purification
Each student or pair of students should have a 10-g
microburger, prepared before class from very lean ground
steak.
3
This sample is placed in a disposable centrifuge tube
and 20 mL (two volumes) of buffer is added. We used 20
mM potassium phosphate, pH 5.6. (Other buffers that can
be used are 100 mM sodium phosphate or 100 mM potas-
sium phosphate, both at pH 7.) Now the student mixes the
sample with a glass rod for 1 min, to break open the cells
and release the Mb, as shown in Figure 2. The student
should be careful: rough or extensive mixing can result in
release of fats and nucleic acids contained in the meat
sample.
Next, to pack the pellet, the sample is centrifuged for
ca. 15 min at 10,000 rpm or for 60 min at 5000 rpm using a
Beckman JA-20 rotor. (The 1-hour time was originally
planned to permit the students to pour and/or equilibrate
their gel filtration columns.) Students should be advised to
balance their tube against another tube, as demonstrated
by the instructor.
After centrifugation, the student will usually observe
a whitish-gray pellet and a reddish supernatant containing
the Mb. This is clear visual evidence that the majority of
the color in the meat comes from the soluble heme protein.
There might also be a top layer of fat, which should be
avoided in pipetting and discarded. The Mb solution (super-
natant) is now carefully removed with a Pasteur pipet. To
prepare the Mb sample for electronic absorption spectros-
copy, the students dilute 1.0 mL of supernatant with 3.0 mL
of the initial buffer.
Electronic Absorption Spectroscopy
The positions of the electronic absorbance bands that
will be studied are listed in Table 1. The basic lab will use
the peaks listed for the visible region, but not those for the
Soret region. The Spectronic 20 single-beam spectrophotom-
eter
1
needs to be warmed up for at least 15 min. The per-
cent transmittance is set to zero by adjusting the left knob.
Next, the buffer is placed in the cuvette (test tube or
Bausch & Lomb cell), and the wavelength is adjusted to 635
nm, the position of a peak found only for the Met-Mb
sample. The absorbance is set to zero by adjusting the right-
hand knob. Next, the diluted Mb supernatant is transferred
into the cuvette, which is placed into the Spectronic 20, and
the absorbance is recorded. At this juncture, it is useful to
teach the students the interrelationship between percent
transmittance and absorbance [A =

log T]. The typical
sample concentrations are easily in the range that can be
accurately determined.
Next, the wavelength is changed to 580 nm and the in-
strument is zeroed with buffer in the cuvette. The student
will now determine the absorbance of the Mb supernatant.
(At 580 nm, only Oxy-Mb absorbs strongly.) This process is
repeated at 542 and 505 nm, being careful to zero the in-
strument with the buffer after each wavelength change.
Students should list both the wavelength used and the
sample absorbance in a table.
Oxidation and Reduction
The remaining Mb supernatant is divided in half. One
half is to be oxidized by thorough mixing with approxi-
mately 15 crystals of potassium ferricyanide (K
6
[Fe(CN]
6
).
This chemical converts Fe
2+
(ferrous) heme proteins to the
Fe
3+
(ferric) form. Here it will produce Met-Mb (sample A),
which is yellow to yellowish brown because of the ferricya-
nide. With the other half of the Mb supernatant, the stu-
dent will mix approximately 20 crystals of sodium dithionite
(Na
2
S
2
O
4
). This is a reducing agent that will convert Fe
3+
heme protein to the Fe
2+
form, producing bright red Oxy-
Mb (sample B). Both samples should sit for 5 min before
absorbance is read.
Electronic Absorption Spectroscopy
Again, samples will be diluted to determine absor-
bance. Use 1.0 mL of sample A (Met-Mb) and 3.0 mL of
buffer to create dilute Met-Mb, and 1.0 mL of sample B
(Oxy-Mb) and 3.0 mL of buffer to prepare dilute Oxy-Mb.
After zeroing the instrument with buffer, students will read
the absorbance for both samples at 635, 580, 542, and 505
nm and make a second table, showing wavelength and ab-
sorbance for dilute Met-Mb and dilute Oxy-Mb. Samples are
expected to be stable for at least 46 hours.
Figure 2. Illustration of procedure for
Mb extraction. The left test tube
shows the meat at the bottom (red-
to-reddish brown), and the buffer
(clear) at the top before extraction of
the protein. After extraction by mixing
with the glass rod and centrifugation,
the buffer (reddish supernatant) now
centrifuge
contains the majority of the Mb, while the meat (pellet) is now
mostly whitish-gray. After carefully decanting the Mb-containing
supernatant, the pellet can be discarded. The extraction of Mb is
now complete.
m o o R p e r P e h t r o f s l a i r e t a M f o t s i L
b a L c i s a B
e b u t e g u f i r t n e c 1
n a m e c i l o p r e b b u r h t i w d o r s s a l g 1
k a e t s n a e l d n u o r g g 0 1
r e f f u b L m 0 5 . a c
h c s u a B 2 & 0 2 c e p S r o f s e b u t t s e t r o s e t t e v u c b m o L
s b l u b d n a s t e p i p r u e t s a P 3 r o 2
e m u l o v L m 0 1 r o 5 , r e d n i l y c d e t a u d a r g 1
s l a t s y r c e g n a r o e n i f , e d i n a y c i r r e f m u i s s a t o p
s l a t s y r c e t i h w " y n i a r g " e n i f , e t i n o i h t i d m u i d o s
d e l l o r t n o c - e r u t a r e p m e t y l b a r e f e r p , e g u f i r t n e c
b a L d e c n a v d A
b a l c i s a b r o f e v o b a d e t s i l g n i h t y r e v e
, e m u l o v d e b L m - 0 1 ~ , n m u l o c x e d a h p e S 5 2 - G d e r u o p e r p
R O n m u l o c y t p m e d n a 5 2 - G n e l l o w s e r p t n e i c i f f u s
r e f f u b l a n o i t i d d a L m 0 5 ~
d e l l o r t n o c - e r u t a r e p m e t y l b a r e f e r p , e g u f i r t n e c
z t r a u q r o , s s a l g , c i t s a l p : e t t e v u c m c - 1
428 Journal of Chemical Education Vol. 74 No. 4 April 1997
In the Laboratory
Advanced Laboratory
Myoglobin Extraction and Partial Purification
This portion of the experiment is identical to that out-
lined in the basic protocol.
Electronic Absorption Spectroscopy
The buffer used to extract Mb from the meat is placed
in a clean 1-cm cuvette. Following directions from the lab
instructor for the particular instrument (scanning spectro-
photometer), a baseline absorbance curve is to be obtained
from 700 to 300 nm.
4
This baseline will be subtracted auto-
matically from the spectrum of the sample when it is
scanned. Next, the supernatant sample is placed in the cu-
vette, and the sample is scanned from 700 to 300 nm. The
sample will contain both Mb-O
2
and Mb-H
2
O and the spec-
trum will show all the peaks listed in Table 1. Students should
label all peaks observed; their spectra can be compared with
those in Figure 3.
This experiment is usually completed in a 2nd lab ses-
sion. The samples should be labeled, covered with Parafilm,
and refrigerated until the next class. At that time, the
samples are removed from the refrigerator. The sample may
have become slightly cloudy owing to the presence of a small
amount of denatured protein. An approximately 10-min cen-
trifugation step using a bench-top centrifuge will enable stu-
dents to separate their meat supernatant (Mb extract) from
the denatured pellet before beginning the next experiments.
Oxidation and Reduction Reactions and Gel Filtration
Chromatography
Column and Sample Preparation
This lab can use columns prepared in advance or pur-
chased, or the students can pour and equilibrate their own
columns. Two chromatography columns, each containing 10
mL of Sephadex G-25 equilibrated in the working buffer, are
required.
5
Next the students will need to equilibrate each
column with 35 volumes (3050 mL) of buffer, if this step
was not already done. (Usually, the students equilibrate
their columns for lab period two while the samples are in
the centrifuge.)
Now the students will divide the Mb supernatant in
half and follow the procedures described for the basic ex-
periment for oxidation and reduction of the two parts of the
sample. The samples should be mixed thoroughly and al-
lowed to incubate for about 5 min. Students should be able
to observe color changes for both samples. The sample to
which the oxidizing agent was added, producing Met-Mb,
will be a yellow-brownish color and the sample to which the
reducing agent was added, producing Oxy-Mb, should be
bright red.
Desalting Experiment
A gel filtration chromatography
5
column with a 10.0-
mL bed volume (V = r
2
h, where V is column volume and h
is column bed height) can efficiently separate 1015% in a
desalting experiment. So an appropriate sample volume
to be loaded is on the order of 1.01.5 mL. Each protein
sample (Met-Mb or Oxy-Mb) is gently loaded onto a sepa-
rate column, being careful not to disturb the gel bed. In the
case of the Met-Mb sample, there will initially be a yellow-
ish-brown band (protein + ferricyanide) that will move down
the column, separating into a lower brown band of MetMb
(ca. 17,000 Da) and an upper yellow band of the oxidizing
agent, which will be slowed down by the beads in the col-
umn. This separation is shown in Figure 4. The brown band
will be collected. To ensure the collection of all the protein,
students can put a white piece of paper behind the column to
better observe the color of the drops as they elute.
In the case of the Oxy-Mb sample, the bright red pro-
tein moves quickly down the column, whereas the colorless
reducing agent is also slowed down by the beads in the col-
umn. The bright red band is to be collected and the students
can use the white-paper method again. An important point
is to collect only the colored drops. This is because although
the sodium dithionite is not colored, if collected with the
sample it will interfere with the spectrum.
Electronic Absorption Spectra
The buffer that was used for the original extraction is
placed into a clean 1-cm cuvette and the instruments
baseline absorbance curve is obtained from 700 nm to 300
nm, as described previously.
4
The samples eluted from the
two columns will most likely be too concentrated to be on
scale in the Soret region. Students can check this by ex-
amining the column eluates at the Soret peaks listed in
Table 1. We usually prepare the sample by adding 2.5 mL
buffer to 0.5 mL of the column eluate. The homogeneous
(post-chromatography) Oxy-Mb and Met-Mb samples are
scanned from 700 to 300 nm. For Met-Mb, the sample
should show all the peaks in Figure 5A; for Oxy-Mb, the
sample should look very similar to the spectrum shown
in Figure 5B.
Discussion
This experiment clearly and visually illustrates several
fundamentally useful biochemical principles: (i) the relative
simplicity of extracting a soluble protein, in this case the
respiratory protein myoglobin, from a tissue in which it is
found at high levels; (ii) the use of redox chemicals and the
generation of different oxidation states for an important bio-
logical system; and (iii) the various uses of absorption spec-
troscopy (e.g., to directly examine an extract, and subse-
quently two different forms of the protein produced from
that extract). It also permits students to directly correlate
their observations of bright red Oxy-Mb with fresh meat
and the brownish color of Met-Mb with old meat.
From the first set of data, students will see that the
Mb supernatant (meat extract) has bands that correspond
to both Oxy-Mb and Met-Mb (Table 1); the complete spec-
trum would closely approximate that shown in Figure 3. In
particular, note that whereas the absorption bands for Oxy-
Mb in the visible region (>500 nm) are essentially distinct
from those for Met-Mb, the strong Soret bands actually
Figure 3. Electronic absorption (UV-vis) spectrum of the meat su-
pernatant. Comparison of this figure with Table 1 reveals that the
supernatant is a mixture of Oxy-Mb and Met-Mb.
Vol. 74 No. 4 April 1997 Journal of Chemical Education 429
In the Laboratory
Figure 4. Diagram illustrating the principle of gel-filtration chro-
matography. (A) Separation of myoglobin from the reducing or oxi-
dizing agent. (B) Close-up illustration of the path of the smaller
molecule (R is the redox agent) into the holes of the Sephadex
gel, while the larger molecule (myoglobin) goes directly through
the column.
A
B
Figure 5. Electronic absorption spectra of (A) Met-Mb and (B)
Oxy-Mb.
A
B
overlap and are not separately resolved. From these data,
students can see that spectra of a mixture contain contri-
butions from each component present. After preparation
and spectral examination of Oxy-Mb and Met-Mb, they will
have data for the visible region corresponding to a homoge-
neous sample of Oxy-Mb or Met-Mb (Fig. 5).
Some Questions for Basic Lab Students
What did you learn that explains why fresh meat is red
and aged meat begins to turn brown?
Think about a different type of meat, like the white
meat vs. the dark meat from turkey. Can you propose a rea-
son for the observed difference in color? [Hint: Why is myo-
globin present in muscle and what function does it serve?]
Some Questions for Advanced Lab Students
Using Beers Law (A = cb), calculate the concentration
of Oxy-Mb and Met-Mb in your samples.
Suppose the 1-cm cuvette had not been available for
your experiments and all you have is a 2-mm cuvette. What
would your absorbance readings have been for the same
samples?
What form of iron, ferric or ferrous, is most readily ab-
sorbed by the body? What does that information suggest
about the dietary availability of iron in aged meat?
Conclusion
These experiments illustrate a variety of useful bio-
chemical methods for students in basic or advanced labora-
tory courses. They also give students an opportunity to do
an experiment on a protein that is not only physiologically
important, but also relevant to their daily lives. This ex-
ample can be seen every time the student goes to the gro-
cery store. The color of meat can now be identified with the
oxidation state of myoglobin, bright red being ferrous oxy-
myoglobin and brown being ferric Met-myoglobin. It is vi-
tally important for students to be able to draw correlations
between their laboratory experiments and lecture discus-
sions and components of their daily lives. Too often, stu-
dents are aware only of the difficulty of doing science. When
connections can be made between the classroom and stu-
dents daily experience, science can more readily become an
important and useful part of their lives.
Acknowledgments
This work was supported in part by a grant-in-aid from
the American Heart Association, #94007920 to L.A.A., and
by the USDA Research Apprenticeship Program for Minor-
ity High School Students. We acknowledge the excellent
work of Isabelle Mitura in initiating this project. The manu-
script is contribution #96-94-J from the Agricultural Experi-
ment Station at Kansas State University.
Notes
1. The Spec. 20 should have either the visible phototube or
the wide-range phototube; the infrared phototube cannot be used.
2. We recommend purchase of a small (about one pound)
lean steak (round steak) the day before the lab experiment. Most
grocery stores will grind this up for you as you wait. One year, we
had a very conscientious student in the prep. room who bought
freshly ground round steak and then used only the bright red por-
tion of the ground meat in preparing the microburgers for the stu-
dents. As a consequence the initial extraction yielded all Oxy-Mb,
and the experimental demonstration that absorption spectra are
additive did not work.
430 Journal of Chemical Education Vol. 74 No. 4 April 1997
In the Laboratory
3. Use of a lower grade hamburger will result in very fatty
preparation, making the partial purification of Mb more difficult.
The 10-g patties can be weighed, wrapped in plastic wrap, and
refrigerated the night before the experiment, or can be prepared
farther in advance and frozen.
4. Although the same baseline could be used for all of the
samples, obviously it is preferable if time permits for each student
to obtain a baseline and scan his or her own sample.
5. Detailed classroom instructions for pouring a chromatog-
raphy column and for gel filtration chromatography are not de-
scribed here because they may readily be found in the literature
(e.g., ref 3).
Literature Cited
1. Ikeda-Saito, M.; Hori, H.; Andersson, L. A.; Prince, R. C.; Pickering,
I. J.; George, G. N.; Sanders, C., II.; Lutz, R. S.; McKelvey, E. J.;
Mattera, R. J. Biol. Chem. 1992, 267, 2284322852.
2. Antonini, E.; Brunori, M. Hemoglobin and Myoglobin in their Re-
actions with Ligands; North-Holland: Amsterdam, 1971.
3. Stellwagen, E. Methods Enzymol. 1990, 182, 317328.