[CANCER
RESEARCH
35, 1505-1509, June 1975]
Characterization and Subunit Analysis of Ferritin Isolated from
Normal and Malignant Human Liver'
Elliot Alpert
Departmentsof Medicine, Harvard Medical School and Gastrointestinal Unit, MassachusettsGeneralHospital, Boston, Massachusetts02114
nant cells in tissue culture and rat hepatoma migrated more
SUMMARY
rapidly electrophoretically
Ferritin was purified from normal, fetal, and malignant
liver tissue. Ferritin purified from hepatoma tissue migrated
slightly faster than normal human liver ferritin in polyacryl
amide gel eiectrophoresis. Hepatoma and fetal liver ferritin
contained an acidic component in gel and liquid isoelectric
focusing not found in normal liver ferritin. We have called it
a carcinofetai
isoferritin. The subunit compositions
of
ferritins purified from human liver cell carcinoma and
normal liver were then compared. Both ferritins consisted of
a subunit species with an identical molecular weight of
approximately
18,500. A single subunit of similar molecular
weight was also demonstrable after dissociation of 8 M urea
and by gel filtration in urea. Two subunits were demonstra
ble in normal liver ferritin by means of acrylamide electro
phoresis in 8 M urea in acid pH. The same two subunits were
also demonstrable in ferritin isolated from human liver cell
carcinoma.
However, a third subunit, intermediate
in
charge between the two normal liver subunits, was demon
strable in different amounts in ferritins from two hepato
mas. Ferritins from normal and malignant livers were
immunologically
indistinguishable.
The tumor-specific
acidic isoferritin was isolated and antisera were prepared.
The isolated acidic isoferritin was found to be immunoiogi
caiiy identical to normal liver isoferritins. It is concluded
that the multiple isoferritins of the human liver ferritin
consist of two subunits, which are identical in molecular
weight but which differ in net charge. Ferritin, isolated from
two human liver carcinoma tissues, was composed of the
same two subunits and a third unique subunit. Different
amounts of these subunits may account for the several
normal isoferritins and a unique tumor-specific acid isofer
ritin found in hepatoma.
INTRODUCTION
An increasing
bodyof evidenceindicatesthat the iron
storage protein, ferritin, is composed of multiple molecular
forms or isoferritins. These isoferritins have been separated
by isoelectric focusing or ion-exchange chromatography
(4,
21, 22). Of particular
interest was the observation
by
Richter and Lee ( 17, 18) that ferritin isolated from malig
1 This
work
was
supported
by
NIH
Grant
CA-12389
and
Cancer Society Grant IM-l4B.
Received October 11, 1974; accepted February 21, 1975.
American
than normal. We have confirmed
and extended these observations by showing that ferritin
isolated from human liver carcinoma
and fetal liver differed
from normal liver ferritin and contained isoferritin variants,
not present in normal liver, which we have called carcino
fetal isoferritins (I). Therefore, in an attempt to character
ize the molecular basis for the additional carcinofetal acidic
isoferritin, we have studied the subunit structure of ferritin
isolated from normal and malignant liver tissue.
MATERIALS
AND METhODS
Fresh tissues were obtained postmortem from the Dc
partment of Pathology, Massachusetts General Hospital,
Boston, Mass., within 12 hr after death. Fetal liver was ob
tamed from therapeutic abortions, performed for medical
legal reasons during early pregnancy. The tissues were
stored at —
20° until the ferritin was extracted, and the
extracted ferritin was kept at 4°
. Ferritin was isolated from
these tissues by the method of Drysdale and Munro (5),
except that carboxymethylcellulose chromatography was
omitted to avoid possible selective loss of isoferritins of
differing charge. The tissues were homogenized and sub
jected to heat and acid precipitation, ammonium sulfate
precipitation,
and final purification by Sephadex G-200
column chromatography
(5). Sephadex was purchased
from Pharmacia Fine Chemicals Inc., Piscataway, N. J.
The purity of each preparation was confirmed by poiy
acrylamide
gel electrophoresis,
a commonly
accepted
presumptive evidence for purity of ferritin. In each case,
only bands that stained for both protein and iron were seen
in positions where ferritin is known to migrate.
Slab electrophoresis
was performed
using a system
purchased from Ortec Inc., Oak Ridge, Tenn. A vertical
quartz cell with internal dimensions of 9.5 cm x 9 cm x 4
mm was filled with 4.5% poiyacrylamide Eastman Organic
Chemicals, Rochester, N. Y. that was water layered to form
a straight edge and then allowed to polymerize. After
removing the water layer, the gel slab was overlaid with 8%
polyacrylamide
gel, containing a Teflon well former, and
allowed to polymerize for 20 mm. After removal of the
Teflon well former, the wells were rinsed with distilled water
and blotted dry. Ferritin samples, purified according to the
identical protocol and containing 50 to 100 @zgof protein,
were each thoroughly premixed with an equal volume of
50% sucrose in 0.0375 M Tris-sulfate, pH 9.0, and placed in
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1505
E. A!pert
the well. Electrophoresis was performed vertically, with the
cathode uppermost, in 0.065 MTris-borate buffer, pH 9.0, at
4° and a constant
voltage
from an Ortec 4l00
pulsed
constant power supply. The pulse rate was increased at
5-mm intervals, from 75 to a maximum
of 300 pulses/sec
at
15 mm. The total running time was approximately 45 mm.
The gel slab was stained for protein using Ponceau S or
Coomassie blue and counterstained for iron with potassium
ferrocyanide. SDS2-polyacrylamide electrophoresis was
performed according to the method of Neville (12). Molecu
lar weights were estimated from precise densitometric
measurements of protein bands compared to standards.
Acid urea electrophoresis was performed in a thin gel slab of
4.5% polyacrylamide
in 6 M urea and 20 mr@iglycine-HCI
buffer at pH 3.0. Purified ferritin preparations were first
dialyzed against 6 M urea at pH 3.0 and then placed in the
wells. The buffer trays contained acid glycine-HCI buffer,
0.2 M in 6 M urea, and electrophoresis
was performed
at a
constant current of 30 ma for about 3 hr.
Preparative isoelectric focusing was performed in a
110-mi sucrose density gradient containing 4% ampholytes
(LKB Productor, Sweden), pH 5 to 7. After 3 to 5 days of
electrolysis, at an initial constant power setting of 2.5 watts
until equilibrium was achieved, 1.0-mi fractions were col
SDS to enhance dissociation
into its subunits (20). They
were then subjected to SDS-polyacrylamide gel electra
phoresis (12) (Chart 2). The gels were stained for protein,
each gel was analyzed by UV densitometry, and migration
indexes were calculated and compared to standards. The
major subunit of each ferritin migrated between myoglobin
and chymotrypsinogen and was calculated by densitometry
to weigh 18,500 daltons. In addition, there were 2 smaller
fragments with molecular weights of approximately 9,000
and 11,000 noted in lower concentration. The same pattern
was reproduced in mixing experiments, where malignant
and normal liver ferritin were mixed and reanalyzed by SDS
eiectrophoresis
showing the major subunit to be indistin
guishable in size by this technique.
Ferritins purified from normal, fetal, and malignant liver
tissue were subjected to preparative isoelectric focusing in a
sucrose density gradient. Quantitative
estimation of the
apoferritin by electroimmunodiffusion
revealed 6 or 7 peaks
in preparation. When the fractions were plotted by the pH
at which they focused as the ordinate (Chart 3), it was
evident that the ferritin from malignant liver had its major
peaks focusing at a pH more acidic than normal. Hepatoma
ferritin had isoferritin peaks common to both normal and
fetal liver ferritin.
However, the single most acidic isoferritin fraction was
lected from the bottom of the column, and the pH gradient
measured with a microelectrode. Apoferritin was identified
and quantitated by electroimmunodiffusion (9) using mono
detectable on subsequent focusing runs (Chart 3). This
specific rabbit anti-human
trofocusing was performed
the isoferritins characteristic of normal liver tissue. This
ferritin. Analytic gel dcc
according to the method of
Righetti and Drysdale (19) in 4.5% polyacrylamide. The gels
were stained for protein with Coomassie blue, immuno
precipitated in situ with anti-liver ferritin antisera (4, 16), or
stained for iron by potassium ferrocyanide. Acid urea gel
filtration was performed in Sephadex G-75 equilibrated
eluted
at pH 5.05, a pH where no normal
fraction was shown by analytical
ferritin
was
gel focusing to be free of
isolated hepatoma specific isoferritin was mixed with equal
volumes of Freund's complete adjuvant and injected into the
with 8 M urea and 0.02 M glycine-HC1 buffer, pH 3.0. The
+
column was calibrated by means of dextran blue, chymo
trypsinogen, and myoglobin standards Pharmacia, Uppsala,
Sweden.
RESULTS
Ferritin
was purified from primary
liver cell carcinoma
and normal adult liver tissue and compared by polyacryl
amide gel electrophoresis in a vertical slab at alkaline pH
(Chart I). All samples were purified according to the same
protocol outlined in “Materialsand Methods―and were
dialyzed against the same barbital buffer, pH 8.6, to
equalize the pH and ionic strength of the samples prior to
electrophoresis. All the bands were stained for both protein
and iron, identifying them as ferritin. Small amounts of
dimer and oligomeric ferritins were also visualized but no
contaminating
non-iron-containing
demonstrating
proteins
were
seen,
the purity of the preparation. The mono
meric form of ferritin isolated from liver cell carcinoma
consistently
migrated slightly ahead of ferritin isolated
from normal liver.
Ferritins
isolated
from liver cell carcinoma,
fetal liver,
and normal liver were incubated at 60°for 1 hr in 0.1%
H
Chart 1. Polyacrylamide
.N
gel electrophoresis at pH 8.6. Ferritin isolated
from hepatoma(H) and normal adult liver (N) subjectedto electrophoresis
after dialysis againstthe samebarbital buffer, pH 8.6, to equalizepH and
ionic strength in the samples. All bands stained for iron and protein with
2 The
I506
abbreviation
used
is
SDS,
sodium
dodecyl
sulfate.
the major band representingthe ferritin monomer.
CANCER RESEARCH VOL. 35
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Norma! and Hepatoma
@.-
suggesting
@
@li!
Isoferritins
lower-molecular-weight fragments previously identified also
appeared in the identical place in most of the peaks tested,
.4-
I@.
Ferritin Subunits,
further degradation
of the major subunit into 2
smaller fragments.
In order to determine whether the major subunit of
ferritin showed variation in net charge, experiments were
performed in 8 M urea in acid pH. The major subunit of
hepatoma ferritin was isolated from its partially degraded
oligomeric forms by gel filtration on Sephadex G-75
equilibrated in 8 M urea, pH 3.0 (Chart 4). The column was
calibrated with dextran blue, chymotrypsinogen, and myo
-MY
-CT
-OA
-BSA
globin.
The
incompletely
denatured
higher-molecular
weights fragments eluted in the void volume. A single
symmetrical peak was seen in the inclusion volume, eluting
just ahead of myoglobin, consistent with a single subunit of
18,500 daltons in urea, and identical to that determined by
SDS gel eiectrophoresis.
Aliquots of ferritin isolated from 2 hepatoma
livers and
H N FStd.
Chart 2. SDS-polyacrylamidegel clectrophoresisof ferritins isolated
from hepatoma(H), normal liver (N), and fetal liver (F). Tracking dye was
marked with India ink to allow precisemeasurementof R@,
. Standards
included myoglobin (MY), chymotrypsinogen(CT), ovalbumin (OA), and
bovine serum albumin (BSA). The major ferritin subunit (arrow) weighed
18,500 daltons.
footpads of 2 rabbits. The rabbits were given boosters at 4
weeks and bled 2 weeks later. The antisera
were tested by
Ouchterlony gel diffusion against the acidic isoferritin used
as the immunizing agent, purified hepatoma ferritin, and
purified
normal
liver ferritin.
A line of complete
identity
was observed with the anti-acid isoferritin antisera against
all 3 ferritin
preparations.
The same result was obtained
using antisera made against purified normal ferritin. No
spurring was noted. Cross-absorption studies were also
performed. Anti-acid isoferritin antisera were absorbed with
normal liver ferritin, and anti-normal
liver ferritin was
absorbed by hepatoma ferritin. After this cross-absorption,
I'
both antisera did not react with any of the ferritin prepara
tions. These data suggest that the tumor-specific acid
isoferritin contains no exposed antigenic determinant that
could be differentiated from normal liver ferritin by the
immunological means used.
The major peaks, as shown in the brackets in Chart 3, as
well as the more acidic and basic fractions on either side,
were pooled from normal, hepatoma, and fetal ferritin after
separation
by preparative
isoelectric focusing. The isolated
isoferritins were dialyzed back to pH 7.4 and
gel isofocusing. The isoferritins redistributed
the characteristic p1 of each isoferritin,
demonstrated (4, 7). Aliquots were dialyzed
reanalyzed by
according to
as previously
against 0.1%
SDS; heated to 60°for 60 mm, and the 3 fractions of each
normal, fetal, and hepatoma ferritin were then compared by
SDS gel electrophoresis. The major subunit, from all
isoelectric focusing fractions in each preparation,
had an
identical molecular weight of 18,500 as measured by
densitometry and calculation of mobility. In addition, the 2
4.9
I
I
I
I
5.1
5.3
5.5
5.7
pH
Chart
3. Preparative
isoelectric
focusing of ferritin
isolated from
normal, fetal, and hepatomalivers in a sucrosedensity gradient using4%
ampholines,pH 5 to 7. Fractions, I .0 ml, were eluted and the pH was
determined. The ferritin protein concentration was measuredimmuno
chemically by electroimmunodiffusion. The main peaks (brackets) were
pooled and comparedto more acidic and basic fractions.
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1507
@
1@
@.V
E. A!peri
demonstrated
that human livers tumors and fetal liver
ferritin contain a unique acid isoferritin band, which we
have called a carcinofetal isoferritin (17). Similar acidic
ferritins have been shown to occur in human pancreatic and
breast tumors (1 1) and in HeLa cells and early placental
extracts (6). Since these multiple forms are not likely to be
due to a single uniform subunit population, we investigated
the subunit structure of ferritins isolated from hepatoma
Sephodu G-75, 8M Ursa
Void volume
Chymo-trypsinoqsn
Myoglobin
80
90
and normal liver.
The molecular weight of horse spleen ferritin subunits
@
I
0
I
10 20
30
I
I
I
I
I
I
40
50
60
70
80
90
I
$00 110
FRACTION NUMBER
Chart 4. Urea gel filtration in SephadexG-75equilibrated with 8 Murea
in 0.02 M glycine-HCI, pH 3.0. The column was calibrated with dextran
blue, chymotrypsinogen, and myoglobin.
several normal livers were then compared by acid urea gel
electrophoresis. The samples were first dialyzed against 8 M
urea in 0.02 M glycine-HCI buffer, pH 3.0, and subjected to
electrophoresis in acrylamide in the same acid urea condi
tions. Two bands (A and B) were consistently noted in
ferritin isolated from normal liver, both migrating slightly
slower than cytochrome c. The same 2 bands were noted in
the hepatoma ferritins although with very different intensi
ties. Furthermore,
there was an additional ferritin subunit
band (C) intermediate between the 2 normal bands, particu
larly evident in the 1st hepatoma (H1) (Chart 5).
The same preparations were also subjected to urea
electrophoresis in 7, 11, and 13.5% polyacrylamide. Electro
phoresis in different polyacryiamide gel concentrations had
no effect on the relative migration of the major subunit
bands (A, B, and C). All additional bands disappeared after
treatment with dithiothreitoi and were also excluded from
the higher gel concentrations. The hepatoma ferritin (Hi)
that had higher concentration of the unique tumor specific
intermediate
band, and less of the normal band B, also
showed a higher concentration of the tumor-specific acid
isoferritin
on gel electrofocusing
(1).
was found to be 18,500 by SDS (2). Niitsu et a!. (13) have
demonstrated subunit heterogeneity in horse spleen ferritin
which we have confirmed (unpublished observations). How
ever, we found only a single major subunit with a molecular
weight of 18,500 in normal human liver ferritin by SDS gel
eiectrophoresis.
We were unable to demonstrate the exist
ence of any major subunit differences in molecular weight in
hepatoma or normal liver ferritin by SDS gel electrophore
sis or by urea gel filtration. However, trace amounts of 2
lower-molecular-weight
fragments were seen in several
preparations of human ferritin, with molecular weights of
approximately
9,000 and I 1,000 by SDS electrophoresis.
These appear to correspond to the lower-molecular-weight
fragments found by Niitsu et a!. (I 3) in horse spleen and
human ferritin.
weight identical
The acidic isoferritin had a molecular
to the basic bands. No additional bands
were detected after careful densitometry measurements, or
by coelectrophoresis.
Gel filtration in urea also demon
strated only a single symmetrical peak in the calibrated
included volume, indicating a molecular weight of about
18,500. Thus, the major subunit size found in urea was
essentially the same as that previously demonstrated
by
SDS gel electrophoresis.
Normal liver ferritin consisted of 2 distinctly different
electrophoretic forms of subunits (A and B) when subjected
to acid urea electrophoresis. The relationship between the 2
subunit bands did not change when analyzed in 4 different
acrylamide concentrations, confirming that the difference
was due to charge differences
only. All slower bands were
removed from normal ferritin by either treatment with
DISCUSSION
Ferritin is an intracellular
iron storage protein that
consists of a hollow protein shell (apoferritin)
in which
inorganic iron may be sequestered (7). It has been thought
that ferritin consisted of 20 or 24 subunits (2, 8), which were
thought to be identical (8). However, multiple molecular
dithiothreitol or $-mercaptoethanol,
as noted by Niitsu and
Listowsky (14), or by the higher concentrations of acrylam
ide. This indicated that the additional components excluded
at higher gel concentrations were aggregates probably
bound by disulfide bonds. Our demonstration of 2 distinct
normal liver ferritin subunits of similar molecular weight,
but differing in charge, may explain the microheterogeneity
@
forms of ferritin, or isoferritins, have recently been demon
strated in horse spleen, rat liver, and human liver by
electrofocusing (1 , 4, 15, 2 1, 22) and ion-exchange chroma
tography (3, 4, 21, 22). These forms have been shown to be
A
@
stable molecular forms unchanged by the pH alterations on
B
@
reanalysis (1, 7). We have previously shown that ferritin
isolated from a human liver tumor has more rapid electro
phoretic mobility than normal liver ferritin (1) and appears
A
N
•
.
A
.
B *@J@
Hi:';@
.
. H.2.
to be similar to the more rapidly migrating “abnormal― Chart 5. Urea gel electrophoresis in 4.5% polyacrylamide equilibrated in
ferritins found in malignant cell cultures (17), the livers of 6 M urea in 0.02 M glycine-HCI buffer, pH 3.0. Ferritins isolated from
tumor-bearing
animals
(10, 18). We have found similar
changes in ferritin isolated from early fetal livers and have
I508
normal liver (N) and hepatoma livers (H, and H2) were dialyzed and
equilibrated in the same 0.02 M glycine-HCI buffer, pH 3.0, and 8 M urea.
CANCER
RESEARCH
VOL.
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35
Normal
of
ferritin
previously
reported
(3,
4,
6,
13,
16,
21,
22).
The
multiple isoferritins, separated by isoelectric focusing or
ion-exchange
chromatography,
could consist of ferritin
shells containing different proportions of the 2 sUbunits.
A 3rd unique subunit (Band C) with an intermediate net
charge has been demonstrated
in the 2 hepatomas tested.
The hepatoma and normal ferritin were immunologically
indistinguishable.
unique antigenic
Nevertheless, the subunits may contain
determinants hidden within the shell and
need to be further characterized.
additional
tumor-specific
The presence of an
subunit in the ferritin shell could
be responsible for the presence of the additional acidic
isoferritin found in human hepatoma ferritin (1). This acid
isoferritin
is present in fetal liver in early gestation,
disappears in late gestation, and reappears in cancer tissue
(1). This suggests that the acid isoferritin and apparently
unique subunit may represent another carcinofetai altera
tion of protein synthesis in tumors.
and Hepatoma
Ferritin Subunits,
Isoferritins
Isoferritin in Placenta and HeLa Cells. Cancer Res., 34. 3352-3354,
1974.
7. Granick, S. Ferritin: Its Properties and Significance for Iron Metabo
lism. Chem. Rev., 38: 379-403, 1946.
8. Hofmann, T., and Harrison, P. M. The Structure of Apoferritin:
Degradation
into and Molecular
Weight
of Subunits.
J. Mol.
Biol., 6:
256-257,1963.
9. Laurell, C. B. Quantitative Estimation of Proteins by Electrophoresis
in Agarose Gel Containing
Antibodies.
Anal. Biochem., 15: 45-52,
1966.
10. Linder, M., Munro, H. N., and Morris, H. P. Rat Ferritin Isoproteins
and Their Responseto Iron Administration in a Series of Hepatic
Tumors and in Normal and Regenerating Liver. Cancer Res., 30:
2231—2239,
1970.
11. Marcus, D. M., and Zinberg, N. Isolation of Ferritin from Human
Mammary and Pancreatic Carcinomas by Means of Antibody Im
munoadsorbents.Arch. Biochem. Biophys., 162:493-501. 1974.
12. Neville, D. M. Molecular Weight Determination of Protein Dodecyl
Sulfate Complexes by Gel Electrophoresis in a Discontinuous Buffer
System. J. Biol. Chem., 246: 6328-6334, 1971.
13. Niitsu, Y., Ishitani, Y., and Listowsky, I. Subunit Heterogeneity in
Ferritin. Biochem. Biophys. Res. Commun., 55. 1134-1 140, 1973.
ACKNOWLEDGMENTS
14. Niitsu, Y., and Listowsky, I. Mechanisms for the Formation of
We wish to gratefully acknowledgethe excellenttechnicalassistanceof
Ruth L. Coston,whosepatienceandperseverancemadethis studypossible.
We also wish to thank Dr. James W. Drysdale for reviewing this
Ferritin Oligomers. Biochemistry, 12: 4690—4695,1973.
15. Powell, L., Alpert, E., Drysdale, J. W., and Isselbacher, K. J. Organ
Specific Forms of Ferritin in Normal Human Tissues and Cirrhosis
and Iron Storage Disease. Gastroenterology, 64: 889, 1973.
manuscript and for his continued interest and collaboration.
16. Powell, L. W., Alpert, E., Drysdale, J. W., and Isselbacher,K. J.
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JUNE 1975
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1509
Characterization and Subunit Analysis of Ferritin Isolated from
Normal and Malignant Human Liver
Elliot Alpert
Cancer Res 1975;35:1505-1509.
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