CLIN. CHEM. 35/5, 778-782 (1989)
Isolationand Characterizationof Myoglobinand Its Two Major Isoformsfrom Sheep Heart
James T. Wu, Robert K. Pleper,
Lily
H. Wu, and Jeffrey L Peters
We isolated myoglobin from sheep heart by homogenizing
cardiac muscle in 70%-saturated ammonium sulfate, followed by chromatography on a column containing carboxymethyl(CM)-Sephadex gel. Two major isoforms of myoglobin, designated Mb 7.9 and Mb 8.1, were separated by
chromatofocusing and were distinguished by their different
patterns seen on either isoelectrofocusing or on electrophoresis on polyacrylamide gel. The isoelectric points of the
major bands of Mb 7.9 and Mb 8.1 were 7.4 and 7.16,
respectively. Both isoforms were identical in size when
examined by gel filtration chromatography but differed slightly when analyzed by polyacrylamide gradient gel in the
presence of sodium dodecyl sulfate. The Mr of Mb 7.9
(15 900 Da) is slightly smaller than that of Mb 8.1 (18 400
Da). When reacted against rabbit anti-sheep myoglobin, two
isoforms also appeared as two nonidentical precipitin lines
on agarose gel.
We used sheep as a large-animal
model to assess the
effectsof mechanical rest and to test pharmacological
theory
concerning
recovery of myocardium
after “stunning”
(1). We
produced the “stunned myocardium”
in the sheep by repeated canal aortic cross-clamps (no coronary flow) and reperfusion (2). A sensitive marker is needed to assessquantitatively the myocardial cellular damage and correlate it with the
biochemical, histochemical,
ultrastructural,
and functional
abnormalities
of the stunned myocardium.
Radioimmunoassay of blood myoglobm (Mb) is known to
be a more sensitive test for acute myocardial infarction
(AMI) than is the measurement of creatine kinase (CK; EC
2.7.3.2), CK-MB isoenzyme, or other markers
(3_8).1 In
general, after myocardial injury, blood myoglobin reaches a
much higher concentration and appears several hours earlier than CK, and it also returns to the normal concentration
more rapidly (4). There is also evidence that the release of
Mb from myocardium appears to be related specifically to
irreversibly damaged or necrotic tissue and not to ischemic
myocardium-as
possibly typified by stunned myocardium
(9). Therefore, Mb is an ideal marker in experimental
animal models because of its sensitivity and rapid response
to tissue injury. Blood myoglobin has also been shown to be
a more sensitive index than CK in assessing the success of
myocardial reperfusion in both man and dog (5). However,
neither antibodies to sheep myoglobin nor kits are currently
commercially available for measurement of sheep myoglobin.
Here we report our success
in the isolation
of sheep
myoglobin
from sheep cardiac muscle and our preparation
in the rabbit
sheep myoglobin and their differentiation
by their different
isoelectric
points (p1), molecular sizes, and imxnunoreactivi-
ties.
Materials and Methods
For various chromatographies,
the G-50 superfine Sephadex gel, PBE 94 gel, PB 96, and carboxymethyl(CM)Sephadex gel and the pre-cast polyacrylamide gradient gel
PAA 4/30 were all from Pharmacia
Inc., Piscataway, NJ.
The bicinchomnic acid reagent for protein determination
was from Pierce Chemical Co., Rockford, IL 61105. Dialysis
bags (Spectralpor
3) with Mr cutoff point of 3500 Da were
from American Scientific Products, McGaw Park, IL 600856787, and were used specifically for diaylzing samples
containing myoglobin.
Other chemicals
such as ammonium
sulfate, monobasic sodium phosphate, glass wool, potassium
ferricyanide, potassium
chloride,
and Ti-is (Trizma) base
were all from Sigma Chemical Co., St. Louis, MO 63178, in
the highest grade available.
For isoelectrofocusing
(IEF), both IsoGel agarose IEF
plates at pH 3-10 and IsoGel isoelectric-point
(p1) markers
were obtained from Hoefer Scientific Instruments, San
Francisco, CA 94107.
CM-S ephadaz gel chromatography.
A 2.5 x 25 cm Kontex
column packed with 105 mL of CM-Sephadex gel was used
for the purification of myoglobin. Phosphate buffer (50
mmol/L, pH 6) at 4 #{176}C
(buffer B) was used for sample dialysis
and column equilibration. After applying the sample, we
eluted the column with phosphate buffer (0.1 mol/L, pH 6.5)
containing 1 mmol of EDTA per liter (buffer A) until two
dark-brown
bands appeared and became well separated
(Figure 1). The chromatography
was done in the cold room
at 4#{176}C,
with the flow rate maintained
at 20 mL/h.
CM Sephadex
U
-0--
Band 2
-
Band 1
of antiserum
to sheep myoglobin.
We also
and characterization
of two isoforms of
report the separation
Departments
of Pathology and Anesthesiology, University of
Utah School of Medicine, Salt Lake City, UT 84132.
1 Nonstandard
abbreviations:
Mb, myoglobin; CM-, carboxymethyl-; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel
electrophoresis;
IEF, isoelectrofocusing;
AFP, alpha-fetoprotein;
and SH, myoglobins isolated from sheep heart.
Received December 15, 1988; accepted February 9, 1989.
778 CLINICALCHEMISTRY,Vol. 35, No. 5, 1989
I
Fig. 1. Appearance of two bands, both containing myoglobin,on CMSephadex column after elution with buffer A
Gel filtration chromatography.
A 2.5 x 70 cm Pharmacia
column packed with 313 mL of Sephadex G-50 (superfine)
was used. Phosphate buffer (10 mmolJL, pH 7) was used for
sample dialysis, column equilibration, and elution. All samples applied were adjusted to exactly 1 mL. This chromatography was all done at 4#{176}C.
Chromatofocusing.
A 1 x 40 cm Pharmacia C-column
packed with 20 mL of PBE 94 gel was used to separate the
isoforms of myoglobin. 2-Ethanolamine
acetate buffer (25
mmol/L, pH 9.4) and a temperature of 4#{176}C
were used for
sample dialysis and column equilibration. Polybuffer (PB)
96-CH3COOH, after 10-fold dilution with distilled water,
was degassed and adjusted to pH 6 at 4#{176}C
and used for
elution. The pH of eluates was measured at 4 #{176}C
and the
absorbance was recorded vs a blank containing
10-fold
diluted PB 96.
IEF. We used an LKB 2103 power supply and an LKB
2117 Multiphor voltage controller (LKB, Gaithersburg,
MD
20877) to perform IEF. The temperature was controlled at
5 #{176}C
by thermostated circulator connected
to a Multiphor
voltage controller. We used commercially pre-made Isogel
agarose plate, pH 3-10. We usually applied a 2- to 3-j.L
sample having a protein concentration of 5 to 10 g/L directly
at the middle of the gel, following the instruction provided
with the commercial film. Three different consecutive constant voltages-e.g., 100 V for 15 mm, 200 V for 1 h, and
finally 1000 V for 20 mm-were applied for the entire run.
SDS-polyacrylamide
gradient gel electrophoresis. We followed the procedure described
by Weber and Osborn (10),
using a commercial pre-made plate (7.5 x 7.5 x 0.27 cm).
The electrophoresis was carried out in the Pharrnacia Gel
Electrophoresis Apparatus GE-2/4. Approximately 10 to 20
g of protein in 2 to 10 tL was mixed with buffer solution
containing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. The mixture was incubated at 45 #{176}C
for 90 mm
before the sample was applied. The gel was pre-electrophoresed at 40 mA for 30 mm. After sample loading, electrophoresis was carried out at 10#{176}C
for 60 mm at a constant
current of 40 mA. The gel was stained with Coomassie
Brilliant Blue R-250.
Preparation of anti-serum in rabbit. We mixed 0.5 mg of
pure Mb 8.1 in 0.3 mL of phosphate buffered saline with an
equal volume of Freund’s adjuvant and used this mixture for
immunization. The rabbits were injected weekly for the first
four weeks, then every third week to maintain the titer.
Blood was sampled from the rabbits weekly between injections.
Polyacrylamide
gel electrophoresis (PAGE). We essentially
followed the procedure
described by Davis (11) but ran the
electrophoresis
on the “Mighty Small I Vertical Slab Unit
SE 200” from Hoefer Scientific Instruments,
using a 75 g/L,
7 x 8 cm gel we prepared ourselves. The electrophoresis
was
carried out at constant current of 30 mA (about 400 V). Each
specimen contained about 5 to 10 tg of protein. The PAGE
took about 40 mm to complete. The gel was then stained
with Coomassie Brilliant Blue R-250.
Isolation Procedure
One frozen sheep heart, approximately
150 to 200 g (wet
weight), was partly thawed at 4 #{176}C.
The tissue was freed of gross fat and connective tissue and
cut into small cubes before mixing with 1.5 volumes of
buffer A containing 70%-saturated ammonium sulfate (buff.
er E) at 4 #{176}C
per volume of muscle tissue.
The muscle was homogenized by grinding at 4#{176}C
in a
Waring Blendor. Several high-speed grindings lasting 30 to
45 s were spaced by 4- to 5-mm soaking periods with the
blender turned off. Grinding was continued until the homogenate was uniform in color and consistency.
The homogenate was centrifuged (19 000 X g, 30 mm,
4#{176}C)
and the supernate was decanted through glass wool.
We oxidized the red solution of oxymyoglobin to fernmyoglobin by adding a 50% molar excessof solid potassium
ferricyanide (approximately 1 g of potassium ferricyanide
per kilogram wet weight of tissue) with gentle stirring at
4 #{176}C
for 1 h. The solution was then dialyzed against buffer B
at 4#{176}C.
CM-Sephadex Chromatoqraphy
We applied at least 2 L of buffer B for column equilibration. Approximately 100 mg of potassium ferricyarnde was
added to the sample before it was applied to the column, to
keep myoglobin in the ferric state during chromatography.
After sample application, about 100 mL of buffer A was used
for elution. Non-heme proteins and excess ferricyanide
usually were eluted from the column. Myoglobin appeared
as two bands; all cytochromes and hemoglobins remained at
the top of the column. At this point, the entire gel was
gently forced out of the column with slight air pressure and
the portion of it containing the two myoglobin bands was
collected in separate beakers. A few milliliters of buffer B
was added to each beaker to make a slurry, and it was
repacked into an 0.8 x 25 cm column and the ferrimyoglobin was then eluted out with Tris HC1 buffer (2 molIL, pH
8.5; buffer C), dialyzed against water, and lyophilized.
Results
Isolation of Myoglobin
The myoglobin so obtained was essentially free of other
proteins, judging from the elution profiles and electrophoretic patterns
of isolated myoglobins (Figures 2-6), after homogenization of the heart muscle with 70% ammonium
sulfate followed by CM-Sephadex chromatography. However, the yield varied considerably from preparation to preparation, ranging from 0.25 to 5.01 mg of myoglobin per gram
wet weight of muscle (Table 1). The yield of myoglobin
usually was higher and the preparation less heterogeneous
when we used either fresh tissues or did the homogenization
while the tissue was still semi-frozen. We included myoglobins recovered from both bands of the CM-Sephadex column
in calculating the yield.
Table 1. isolation of Myoglobin from Sheep Heart (Eight
Preparations)
Tissue,
Myoglobin
Ratio
mgb
mglg#{176}
218
100
0.46
5.3
166
48
0.29
5.0
5.2
270
67
0.25
126
216
1.71
5.4
115
53
0.46
5.4
126
46
0.36
4.4
192
963
5.01
5.5
200
114
0.57
5.2
aWet weightsof the heart tissue after fat and connectivetissue removed.
ga
b
Myoglobin Concentration based on protein determination with bicinchoninic
acid reagent (Pierce) and bovine serum albumin was used as standard.
C Amount of myoglobin isolated expressedas pergramwet weightof heart
tissue.
CLINICALCHEMISTRY, Vol. 35, No. 5, 1989 779
G-50
We identified myoglobmnbased on three of its properties:
its relatively small molecular size (approximately 17 200
Da), its relatively basic isoelectric point (>7), and its high
409/280 nm absorbance ratio (12, 13). This combination of
properties easily distinguishes myoglobin from other protein contaminants during the purification process. The
spectrum
(below in Figure 7) for the purified myoglobin also
suggests that the preparation was practically free of any
protein contaminants.
Vt
O
whale
U
Two Major isoforms by Chromatofocusing
The technique of chromatofocusing allows the separation
of proteins based on their differences in p1. Because the p1 of
myoglobin is basic, we used chromatofocusing to remove
contaminating proteins from the myoglobin isolated by CMSephadex chromatography. Instead of finding any contaminants (Figure 2) we found more than one peak of myoglobin
in the elution profiles of chromatofocusing. We believed that
the myoglobins represented by the two peaks are isoforms,
because these peaks were all eluted at relatively high pH
and all had the same molecular size as sperm-whale myoglobin (Figure 3) and a high 409/280 nm ratio of absorbances.
However, the pH at which these peaks were eluted was not
exactly reproducible from one chromatofocusing to another.
Many variables may affect measurement
of the pH of the
eluates. When the pH meter was carefully standardized and
the pH of the eluates was measured at 4#{176}C,
we found that
the more acidic peak was eluted at pH 7.9 (isoform Mb 7.9),
the other peak at pH 8.1 (isoform Mb 8.1).
0
.0
0
0
.0
50
100
150
200
250
300
3
0
LL
Sheep
-0--
j
Band I & 2
0.4
409 nm
280 nm
-.--
0.3
0.2
0.0
0.1
50
#{149}
p
100
150
200
250
300
30
Volume (mL)
Fig. 3. Gel-filtration chromatography of myoglobins on a column
containing G-50 (superfine) Sephadex gel
Myoglobineluted from either band 1 or band2 of the CM-Sephadex
column
evkienhlyis the same size as sperm-whalemyoglobin,which is known to be
17 500 Da
Further Characterization of the Two Isoforms
75%
PAGE
SH
8.1
SH SH
7.9 8.1
+
7.9
(-)
We further characterized these two isoforms of myoglobin
by gel filtration chromatography (Figure 3), PAGE (Figure 4),
isoelectrofocusing (Figure 5), and by use of a 4-30% SDSpolyacrylaimde gradient gel (Figure 6). As shown in Figure
4, the Mb 8.1 apparently has a different pattern from that of
Mb 7.9. We also found, in a separate PAGE run (Figure 8),
that Mb 8.1 and alpha-fetoprotein appeared at the same
position, even though the myoglobin has a much more basic
p1. The two isoforms also showed two different IEF patterns
(Figure 5). By comparison with the protein markers of
known p1, p1’sof 7.16 and 7.4 were assigned to Mb 8.1 and
Mb 7.9, respectively (Figure 5).
Gradient SDS-PAGE demonstrated that the two isoforms
differed slightly in size (Figure 6). Apparently, gel filtration
on a column containing superfine G-50 was not as sensitive
as gradient SDS-PAGE in making this distinction. By com-
(+)
Fig. 4. PAGE patternsof the two isoforms of myoglobinMb 8.1 and Mb
LI
0
0
0
U,
0.
.0
7.9 (SH 8.1 and SH 7.,
showing differentelectrophoreticpatterns
parison with protein standards
of known molecular mass we
estimated the molecular mass of Mb 7.9 (SH 7.9 in Figure 6)
to be 18400 Da and that ofMb 8.1 (SH 8.1 in Figure 6) to be
15 900 Da.
Nonidenticai Precipitin Lines
10
20
lube
30
40
50
Number
Fig. 2. Elutionprofiledemonstratingtwo isoforms of myoglobin separated by chromatofocusing
780 CLINICALCHEMISTRY,Vol. 35, No. 5, 1989
We prepared anti-sheep myoglobin by immunizing
the
with Mb 8.1, because by electrophoresis
it appeared
to be less heterogeneous. The non-identical precipitin lines
formed between the two isoforms and anti-myoglobin
antibody on agarose gel in the Ouchterlony technique (Figure 7)
indicate that the two isoforms also differ in their immunorerabbits
I EF
6.2.7.
6.0
a3.6’.’
p1
Markers
4
511
511
Mb
0.1
SH
81’79
Fig.7. Doublediffusionbetweenanti-myoglobin
(centralwell)antiserum
and Mb isoforms
Olstance
(crc)
Fig.5. IEF patternsof two isoforms of myoglobin
Mb 8.1 (SH8.1) and Mb 7.9 (SH 7., showingdifferentIEF patterns.The major
bands of two isoforms are marked by amws. Albumin (A/b) was used as a
control.(Below)Determinationofp1forMb 8.1 andMb 7.9 bycomparingwithp1
markers.The p1of Mb 7.9 is7.4 and of Mb 8.1, 7.16
SDS-PAOE, 4-30%
Anti-Mb 8.1 antisewmreacts with both Mb isoforms (Mb 7.9 andMb 8.1) forming
nonidenticalprecipitin lines between two isotorms. BI, 0.2 gIL; 82, 0.1 g/L; Al,
0.2 g/L; AZ 0.1 g/L; A3, 0.25g/L; A4, 0,05 g/L
only a single band was associated with myoglobin
derived from band 2 (Figure 8). Moreover, when we compared the patterns of Figures 4 and 8 we found that the
myoglobin derived from band 2 is actually Mb 8.1 (Figure 4,
SH 8.1) whereas the myoglobins eluted from band 1 (Figure
8, SH B1) are a mixture of both isoforms of myoglobin
(Figure 4, SH 8.1 + 7.9).
whereas
Discussion
...__.
(.r)
6(14,400)
$11 SHSHM
79
7951
a.
We found the isolation procedure
originally
designed for
sperm-whale myoglobin (12) to be equally useful for isolating myoglobin from sheep heart. Results by both gel filtration chromatography and chromatofocusing indicated the
absence of major contaminants
in our final myoglobin
preparation. Apparently, no additional purification step is
necessary after CM-Sephadex column chromatography.
We sought to ascertain whether the two bands of myoglobin that appeared on the CM-Sephadex column and the
materials represented by the two myoglobin peaks isolated
from chromatofocusing were related to problems of purity or
were in fact different myoglobins. We believe that they are
7.5% PAGE
S
E
(-)
I.
015
035
055
075
005
Fig. 6. Determinationof Mr for
myoglobin isoforms by SDS-gradient
4-30%
(Tq,) Mb 8.1 (SH 8.1) evidentlyis slightly largerthan Mb 7.9 (SH 7.. Mr,
molecular-mass standards:phosphorylase(1), albumin (, ovalbumin (, carbonicanhydrase(, trypsininhibitor(S) and alpha-lactalbumin(5).Only the Sizes
of trypsin inhibitor and alpha-lactalbumin are listed (in parentheses).
(Bottom)Judgingby the comparisonwith standardsof known moleculermass,
PAGE,
theM,ofMb8.1
is l8400DaandthatofMb7.9isl5900Da
activities. We believe that this difference in iminunoreactivity will eventually lead us to the development of monospecific polyclonal antibodies and probably a specific assay for
their distinction.
Band 1 and Band 2, CM-Sephadex Chromatography
As noted earlier, two bands of myoglobin appeared on the
CM-Sephadex column when it was eluted with buffer A.
Guided by both the elution proffles of Figure 9 and the PAGE
patterns of Figure 8, we found that the myoglobin associated
with band 1 frequently appeared to be more heterogeneous,
(+)
SH
SH
Bi
B2
AFP
Fig.8. PAGE analysisof band1 (B1) andband2 (B2) of CM-Sephadex
column
Myoglobinsof sheep heart (Sfr’ recovered from band 1 and band 2 of CMSephadexcolumn (see Figure1) havedifferentPAGE patterns.Alpha-fetoprotein
(AFP) was used as a control
CLINICAL
CHEMISTRY,
Vol. 35,
No. 5, 1989
781
30
0
20
40
00
60
5000
0
10
20
30
40
60
Tube
Number
Fig. 9. Demonstrationthat band 1 and band 2 myoglobinsrecovered
fromCM-Sephadexcolumnare usuallyassociated with two peaks and
one peak, respectively,by chromatofocusing
isoforms of myoglobm, because we have characterized
these
molecules by determining
their molecular
size by use of both
gel filtration chromatography and gradient SDS-PAGE; by
investigating their charge properties with chromatofocusing
and IEF; and by measuring their absorbances at 409 and
280 mrs. Both molecules fulfil the three above-mentioned
criteria for myoglobin, although they differ slightly from one
another in all three properties.
The proportion of the myoglobins appearing in the two
bands on the CM-Sephadex column was not constant from
preparation to preparation; we do not know why. Nor do we
know why band 1 or the fast-moving band on CM-Sephadex
frequently contained both isoforms, whereas band 2 was
always associated
with only Mb 8.1. The p1 of Mb 8.1 is 7.12
and p1 ofMb 7.9 is 7.4, so we would expect Mb 8.1 to be less
positively charged at pH 6.5 (the pH of the elution buffer)
than Mb 7.9 and hence to be eluted faster from a cationexchange column such as a CM-Sephadex column. However,
the results we obtained on CM-Sephadex chromatography
were exactly the opposite. Perhaps bindings between the
myoglobins and other unknown ligands caused myoglobin to
deviate from its usual chromatographic behavior.
Similarly, we do not understand why Mb 8.1 was eluted at
a more alkaline pH than Mb 7.9, even though it has a lower
isoelectric point. We would expect Mb 7.9 to be eluted at a
higher pH, because its p1 is 7.4, and Mb 8.1 to be eluted at a
lower pH, because its p1 is 7.12. In summary, the migration
of two isoforms observed in PAGE agrees with the prediction
based on their different isoelectric points. On the other
hand, the order of appearance of the isoforms is the same in
both CM-Sephadex chromatography and chromatofocusing
but is exactly opposite to what we expected from the charge
properties they displayed on electrophoresis.
Therefore, we recommend that the more precise and
reproducible IEF or PAGE patterns should be relied upon for
definitive identification or for differentiation between myoglobin isoforms. Even though we depend on the technique of
782 CLINICALCHEMISTRY,Vol. 35, No. 5, 1989
chromatofocusing to separate myoglobin isoforms, the procedure simply has too many variables to allow accurate
routine identification or characterization of isoforms. Calibration of the pH meter, temperature control during the pH
measurement, column elution rate, and freshness of the gel
all affect the pH of the myoglobin peaks.
The identification of two major isoforms of myoglobin
suggests various research possibilities. The different immunoreactivities of two isoforms also provide the potential
for us to prepare monospecific polyclonal or specificmonoclenal antibodies for the development of a sensitive and specific
immunoassay. In many respects the isoforms of myoglobin
are similar to various isoenzymes. Possibly various tissues
contain different isoforms or different proportions of the two
isoforms, and this may be potentially useful in differentiating between different tissue injuries, e.g., cardiac injuries
from damages to skeletal muscles. Determination of Mb
isoforms may also provide a better diagnosis for various
muscle diseases (13). Because pure Mb isoforms are available, we can see whether they have different
affinities
for
oxygen and various ligands. Such information may be
physiologically important, e.g., may explain their different
distribution in various tissues.
This work was supported in part by research grants ROlHL33805-03, R01-HL31215-02, and R44-HL34283-01 from the National Institutes of Health.
References
1. Rich GF, Smith KW, Murashita
J, GindorfJ, Crofts J, Peters JL.
Ischemic dysfunction: relationship to mechanical rest. Trans Am
Soc Artif Intern Organs 1983;29:84-9.
2. Kloner BA, de Boer LWV, Darsee JR, et al. Prolongedabnormalities of myocardium
salvaged by reperfusion.
Am J Physiol
1981;241:H591-9.
3. Venge P. Does serum myoglobindeterminationhave a place in
the early diagnosis of acute myocardial infarction? Practical Cardiol
1985;11:69-79.
4. Cairns JA, Missirlis E, Walker WHC. Usefulness of serial
determinations of myoglobin and creatine kinase in serum compared for assessment of acute myocardial infarction. Clin Chem
1983;29:469-73.
5. Ellis AK,
Little T, Xaki Masud AR, Klochke FJ. Patterns of
myoglobin release after reperfusion of injured myocardium. Circulation 1985;72:639-47.
6. Johnson RN, Neutze JM, Kerr AR, Gillain B. Serum myoglobin
concentration as an index of myocardial damage after cardiac
surgery. hit J Cardiol 1983;4:33-47.
7. Stone MJ, Waterman MR, Harimoto D, et a!. Serum myoglobin
level as a diagnostic test in patients with acute myocardial infarction [Abstract]. Br Heart J 1977;39:375.
8. Maddison A, Craig A, Yusuf S, Lopez R, Sleight P. The role of
serum myoglobin in the detection and measurement of myocardial
infarction. Clin Chim Acts 1980;106:l7-28.
9. BlockMl, SaidJW, Siegel RJ, FishbeinMC. Myocardialmyoglobin following coronary artery occlusion. An ininiunohistochemical
study. Am J Pathol 1983;1l1:374-9.
10. Weber K, Osborn M. The reliability of molecular weight
determinations by dodecyl sulfate-polyacrylamide
gel electrophoresis. J Biol Chem 1969;224:4406-12.
11. Davis BJ. Disc electrophoresis-ll:
method and application to
human serum proteins. Ann NY Acad Sci 1964;121:404-27.
12. Hapner KD, Bradshaw RA, Hartxell CR, Gurd FR. Comparison
of myoglobins from harbor seal, porpoise and sperm whale. J Biol
Chem 1968;243:683-9.
13. Poche H, Hopfenmuller W, Hoffmann M. Detection and identification of myoglobin in serum by immunoblotting. Effect of exercise
on patients with Duchenne muscular dystrophy. Clin Physiol
Biochem 1987;5:103-11.