Electrophoretic Studies on the Soluble Proteins from
Livers of Rats Fed Aminoazo Dyes*
SAM SOROF,t
PHILIP
P. COHEN,
AND J. A.
(Laboratory of Physiological
of Miller,
Miller,
(1, 3, ,5, 7—10)on the occurrence
MadisOn,
and co-workers
as long as the animals
are maintained
METHODS
on
the dye. However, the protein-bound
dye cannot
be detected in the aminoazo dye-induced tumors1
and this lack can be interpreted as indicating a
qualitative difference between the proteins of liver
and those of the tumors.
Over one-half of the bound dye in these livers is
combined with the soluble proteins (7—10).The
present investigation was undertaken to ascertain
the electrophoretic group(s) of the soluble rat liver
proteins which were combined with the dye follow
ing the ingestion of aminoazo dyes that have dif
ferent carcinogenic activities. In previous studies
(12, 13), it was found that the soluble proteins
of
normal rat liver, of livers from rats fed 4-dimethyl
aminoazobenzene
for varying lengths of time short
of neoplasia, and of liver tissue adjacent to DAB
induced hepatomas
had similar electrophoretic
patterns. On the other hand, the soluble proteins
of DAB-induced hepatomas, their metastases, and
a group of other tumors showed electrophoretic
patterns
which were very similar
to each other
Wi,.)
hibited a striking decrease in the slow moving h
components and contained more of the A and, to a
lesser extent, the N components. It will be shown
in the present report that the major portion of the
protein-bound
azo dye migrates electrophoreti
cally with the same h components (probably h1)
which were previously found to be markedly re
duced in the DAB-induced tumor (13).
of protein-bound
dye in the livers of rats fed the hepatic carcinogen
4-dimethylaminoazobenzene
(DAB) or certain of
its derivatives have directed attention to the pos
sible causal role of these proteins in aminoazo dye
carcinogenesis. The protein-bound
dye appears in
the liver a few days after the initiation of dye-feed
lag and is found in the non-neoplastic
portions
throughout
the stages of tumor formation and
growth,
MILLER,
Chemiatry, and the McArdle Memorial Laboratory, the Medical School, University
of Wisconsin,
The studies
E. C.
MILLER
but
different from these livers. In contrast to the non
neoplastic liver tissues, all the tumors studied ex
Male rats of the Sprague-Dawley
strain, weigh
ing 175—220gm., were fed ad libitum a semi-syn
thetic diet (2, diet 3). The aminoazo dyes were
incorporated into the diets at a level of 0.058 per
cent for 8'-methyl-4-dimethylaminoazobenzene
(3'-Me-DAB) and 2-methyl-4-dimethylaminoazo
benzene (2-Me-DAB),
and 0.064 per cent for
4'-methyl-4-dimethylaminoazobenzene
(4'-Me
DAB).
aAidedin partby research
grantsfromthe WisconsinBy
Alumni Research Foundation, the National Cancer Institute
of the National Institutes of Health, Public Health Service,
and the American Cancer Society. The generousand valuable
The
diet
with
3'-Me-DAB
contained
1.0 mg riboflavin/kg,
while the other two diets
contained 2.0 mg/kg. Two rats from each group
were killed for each analysis. Two analyses were
made on the livers from four rats fed 3'-Me-DAB
for 2.5 weeks, two on the livers from four rats
fed 4'-Me-DAB for 8 weeks, and one each on the
livers of two rats fed 2-Me-DAB for 11, 14, or 16
weeks. At these times the levels of bound dye
were approximately
at maximum (3). For each
analysis the soluble proteins were prepared with
a Potter-Elvehjem—type
lucite homogenizer and
electrophoretically
analyzed,
by the technics
previously
described
(11—13). After the pat
terns were photographed,
the bottom cell see
tion was closed off to isolate the two vertical limbs.
means of a 5-mi. syriiige,
inch, 20-gauge stainless
to an adjustable
equipped
with a 15-
steel needle and attached
screw rack, fractions
throughout
the entire cell were removed in the order of the
assistance
rendered by Mrs. Betty F. Claus is gratefully
Roman numerals shown in Chart 1. The volumes
acknowledged.
t Present address, National Cancer Institute, Bethesda, were noted, and the entire samples were trans
ferred quantitatively
to tared 15-ml. centrifuge
Maryland.
tubes. The protein was precipitated
with tri
Received for publication January 8, 1951.
388
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Cancer Research
384
chloroacetic acid at a final concentration
of 9 per
cent. The sedimented protein was washed once
with 1 M, pH 5 acetate buffer and extracted 4
times for 5 minutes each with 6 ml. of ethanol at
750
C.
After
the
protein
samples
were
dried
in
extracted in the above manner agreed closely with
analyses on larger samples of the same liver pro
tein, which had been subjected
to exhaustive
extraction
with hot ethanol as previously de
scribed (1, 3).
vacuo over CaCl2 and finally over 112504, they were
weighed and analyzed for total bound dye (1, 3).
Bound dye analyses on similar weights of protein
z
. @b
.?@
RESULTS
As previously found with DAB (13), the soluble
liver proteins from rats fed 3'-Me-DAB,
4'-Me
DAB, or 2-Me-DAB for periods of time short of
hepatoma
development
yielded electrophoretic
patterns (Table 1) similar to those for normal liver
(12). In most cases light yellow boundaries were
observed with unfiltered light to move with the h1
component. In two anomalous runs this light yel
low boundary migrated with the h2 component (see
footnote of Table 1). These yellow fronts have not
been seen in preparations
from
rats not fed dye.
tionation of the soluble liver proteins from rats fed aminoazo
In agreement with previously reported results
(7—10),all the soluble protein preparations
con
tamed high concentrations
of bound dye. Chart 1
depicts the mapping of the pipetted electrophoretic
fractions, while Table 2 shows the concentrations
of total bound dye per milliliter for two representa
tive experiments with 3'-Me-DAB. The dye con
tents are expressed in E values, i.e., the optical
densities of acid solutions of the dyes obtained fol
dyes.
lowing
>
z
@0
.—
CHART 1.—The
typical
mapping
of an electrophoretie
frac
TABLE
ELECTROPHORETIC
ANALYSIS
extraction
hydrolyzates
of the
1
OF THE SOLUBLE LivER
PROTEINS
FROM RATS FED
AMINOAZO
2-Me-DAB
11
2
1
2-Me-DAB
14
2
1
3'-Me-DAB4'-Me-DAB2.584422
Dye fed
Weeks fed
No. of rats
of alkaline
No. of analyses
DYES
2-MeD-AB
16
2
1
N
A
—7.25
(7.13—7.40
Momunte
)c:1o-' cm'/volt see.
—6.85—7.06—7.27—5.90—6.15—6.05—4.98—5.10—4.83—3.99—8.8
—7.19 (7.16—7.21
—6.28 6.08—6.30
—6.04(6.03—6.05
as
b
—4.98 4.95—5.04
—3.95 3.89—4.02
—3.14 8.06—8.22
—2.362.23—2.48
—1.711.63—1.75
—4.68 (4.61—4.74)
—3.64(3.60—3.68)
—2.82 (2.76—2.88
—1.00 0.98-1.02
—1.120.90—1.33)
—0.34 (0.26—0.88)t
+0.87 (0.71—0.96)
+1.08
Components
@
@
h,
—2.05
(1.98—2.12)
•—2.05
—1.462
—1.27—0.86—0.96—0.78—0.84—0.17t—0.18
—0.42 0.80-0.53)
0.92—1.23)
Aaza
(per cent)
N
A
a@
a@
b
as'
20.8
19.6
3.7
(2.5-5.0)
7.9
(5.2—10.0)
h2
i
5.2(3.9-6.1)t
1.7(1.4—2.1)
electrophoretic
data
of preneoplastic
20.1—21.5)
18.7—20.4
12.3 (12.0—12.6
7.3(6.0-8.3)
ng@
4.74.13.29.56.79.019.819.420.021.525.718.211.49.214.812.313.53.5.3.68
8.9 8.1—9.7)
9.9(8.4—11.3)
29.1(24.0-31.7)
17.5 (16.8-18.9)
9.5 (8.5-10.2)
ill
S Descending
2 . 6 (2.0—3.2)
6.2(3.4—9.5)
12.1 (10.0—14.1)
12.5t
7. 7t
2.1(1.3-2.9)
liver
supernatants
of rats
fed
3'-Me-DAB,
4'-Me-DAB,
and
5-Me-DAB.
t These data have been directly affected by a spiking of the descending h, component (cf. Ill). Where more than one deterininatk@nis avail
able, suchfiguresare omitted.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1951 American Association for Cancer Research.
SOROF
et al.—Electrophoreiic
Studies
proteins (1). The values E (hi-corrected)/ml
for
the border fractions (II and X) were calculated by
correcting the concentrations,
E/ml, of the bound
dye to take into account the dilutions caused by
the relative electrophoretic position of the h1 corn
ponent within these fractions.
This corrected
value would be the approximate
concentration,
on Protein-bound
385
Dyes
boundary (fractions VI, XI, and XII) as 100 per
cent, the E (h1-corrected)/ml
value of the border
fraction (II) can be used to calculate the per
centage of the bound dye migrating with the h1
component.
Another method of calculation,
by
difference, involves the determination
of the per
centage of the bound dye (E/ml of VI, XI, and
TABLE
2
THE CONCENTRATIONS OF PROTEIN-BOUND DYE IN THE ELECTROPHORETIC FRACTIONS OF THE
SOLUBLE LIVER PROTEINS FROM RATs FED 3'-METHYL-4-DIMETHYLAMINOAZOBENZENE5
FRAcTIoNS
No.
1
Uwi@s
E/ml
E(le@)
11th
I
0
H
0.09
III
0.13
IV
0.16
V
0.17
VI
0.13
XII
0.18
XI
0.17
U
a
a
a
a
a
IX
0.04
VIII VII
0.05 0
a
a
U
0.14
0
2.44
1.91
0.98
0.81
0.74
0.75
0.61
0.64
0.26
0.11
0
E/mI
E(i,-@) /5th
E
0
0.09
0.19
0.18
0.20
0.21
a
o.is
a
a
a
a
ca.
0.20
0.22
a
0.13
0.15
0.01
a
0.01
a
0
a
100 mg. protein
0
2.22
ca.
0.56
0.63
0.60
0.38
0.14
0
E
100 mg. protein
2
X
0.14
0.16
S Concentrations
of protein.bound
aso dye
in the
2.41
pipetted
0.97
0.77
electrophoretic
0.72
fractions
of the
a
soluble
liver
proteins
of rats
fed
3'-Me-DAB.
XII) which is moving faster than the h(h1 + h2)
components on the ascending side (fraction IX).
Ignoring the epsilon boundary and minor i corn
ponent (especially since the yellow boundaries
moved with the h1 component), the remainder is
considered to migrate with the h (probably h1)
components. A sample calculation for experiment
No. 2 of Table 2 is shown below.
E/ml, in the border fractions, if they could be
pipetted in such a way as to exclude portions of the
cell not containing proteins of the h(h1) compo
nents. It is seen that the protein-bound
azo dyes
were present at high concentrations
in fractions
il—VI and X—XII.
Considering the range of E/ml values for the
fractions of the cell containing protein without any
a) Moving with h components (calculated as h1):
E (hrcorr) /rnl of fraction II
E/mloffractionXll
E (hrcorr) /rnl of fraction II
E/ID.1 of fraction XI
0.15
x 100=
0.20
0.15
75percent.
@i]@ X 100 = 68 per cent.
b) Moving faster than h components on ascending side:
E/ml of fraction IX
E/ml of fraction XII
E/ nil of fraction IX
E/m! of fraction XI
On the latter basis, 95 per cent of the total bound
dye in the supernatant fluid moved with a mobility
slower than that of the electrophoretic components
in fraction IX. This slower mobility corresponds
to that of the h components, again ignoring the
salt and minor i boundaries. Therefore, as the over
all range, 68—95per cent of the protein-bound
dye
derived from 3'-Me-DAB
and contained in the
supernatant
fluid moved with the h components
(probably h1). Table 3 contains data obtained in a
similar manner from the experiments performed
with the soluble protein-bound dyes from the livers
0.01
0.20 X 100 = 5 per cent.
0.01
0.22 X 100 = 5 per cent.
of rats fed 3'-Me-DAB, 4'-Me-DAB, and 2-Me
DAB.
Similar conclusions are also reached by calcu
lating the data in terms of bound dye per unit
weight of protein. As shown in Table 2 for two ex
periments with 3'-Me-DAB, the proteins in frac
tions II and UI (composed largely of the h corn
ponents) contained the highest concentrations
of
bound dye. The values then decreased progres
sively through the fractions clockwise around the
cell, as shown in Chart I.
Therefore, if the protein-bound
dye is consid
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Caiwer Research
886
ered to be migrating with the h1 component, the
observed electrophoretic mobility (in veronal buff
er, 0.] ;@,at pH 8.6) of the majority of the soluble
proteins
bearing
the dye was —0.98 X 1O@
cm'/volt sec. (average of all analyses in Table 2).
The mobility value for the corresponding
corn
TABLE
3
THE RELATivE AMOUNTS OF THE SOLUBLE
PROTEIN-BOUND DYEs MIGRATING WITH
THE h COMPONENTS FROM THE LivERs OF
RATS FED AMINOAZO DYEs5
Carcinogenicity
Dye fed
(4)
Per cent of protein
bound dye mi
grating with the
h componentst
10—12
3'-Me-DAB
69—78
68—95
61—78
<I
4'-Me-DAB
55-86
0
2-Me-DAB
S The
per
esnt
of
the
total
43—100
67—100
46—100
soluble
protein.bound
migrating with the h components for rats fed 3'.Me-D
4'-Me-DAB,
and 5-Me-DAB
in the indicated diets.
t Each rangerepresentsthe values obtainedbj the two
methods of computation for each experiment. The lower
figure was obtained by calculation (a) and the higher figure
by calculation (b) as described in the text.
ponent in normal rat liver (13) was previously
found to be —0.99 ±0.09' X 1O@ cm2/volt sec.
If the dye is considered to be carried by the pro
teiiis of the two h components (hi + h2), the range
of similar values would be —0.32 to —0.98 X
10-6
cm2/volt
sec
(Table
2).
The
corresponding
range of values for normal rats (18) is —0.35 to
—0.99
± 0.10
X 1O—@
cm2/volt
sec.
DISCUSSION
Miller and Miller and associates (1, 3, 6) have
suggested that the aminoazo dyes might induce
liver tumors by reacting with certain essential
liver proteins (for example, those required for the
control of growth) in such a manner that cells
might arise which lacked these proteins. The lack
of protein-bound
dye in the tumors produced by
the aminoazo dyes was one of the major facts
which led to these general considerations.
The
findings in the present communication
are in har
mony with this protein depletion mechanism of azo
dye carcinogenesis in that the proteins of the elec
trophoretic
group, which include the aminoazo
dye-protein(s),
are those greatly
reduced
in
amount
in liver tumor
(12). Of great
general
inter
est is the observation that the same components
are also low in a variety of other tumors (12).
@
1 Standard
deviation
=
I@(d2)
That the proteins combined with derivatives of
3'-Me-DAB, a very potent carcinogen, have the
same electrophoretic
mobility as those combined
with derivatives of the very weak carcinogenic
dyes 4'-Me-DAB and 2-Me-DAB indicates that
(a) the three aminoazo dyes give rise to the same
azo dye-containing protein(s) of the h components
or (b) electrophoresis under these conditions can
not detect the existent differences between the
protein(s) bound to the derivatives of these three
azo dyes. It is possible that other conditions of
electrophoresis or other methods could distinguish
between these proteins. In any case, considerable
differences in the rate of accumulation
of these
protein-bound
dyes exist in vivo and can be cor
related with the carcinogenic
activities of the
parent dyes (3).
The finding that the soluble proteins bound to
the azo dyes migrate with the protein(s) of the h
components does not necessarily mean that pro
tein(s) of the h components from normal rat liver
are bound to the azo dyes. Conceivably, soluble
protein(s) of other electrophoretic
components, or
even insoluble proteins, may act as precursor(s) of
these observed aminoazo dye-protein(s)
that have
the mobility range of the h components. In any
case, the finding that the greater portion of the
protein-bound
dye travels with a small fraction
(Table 2; normal rat liver [13] : h1 = 8.0 per cent,
and h1 + h2 = 14.7 per cent) of the total soluble
liver proteins suggests that mekibolü@
derivatives of
these aminoazo dyes bind specific protein(s).
SUMMARY
1. The soluble proteins from the livers of rats fed
3'-methyl-4-dimethylaminoazobenzene,
4'-methyl
4-dimethylaminoazobeuzene,
or 2-rnethyl-4-di
methylarninoazobenzene
were subjected to elec
trophoretic analysis. In each case the major por
tion of the protein-bound
dye migrated with the
slowly migrating h components.
These compo
nents were previously shown to be present in
greatly reduced quantities
in azo dye-induced
hepatomas.
2. The results are interpreted to indicate that
metabolic derivatives of these aminoazo dyes bind
specific liver protein(s).
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Electrophoretic Studies on the Soluble Proteins from Livers of
Rats Fed Aminoazo Dyes
Sam Sorof, Philip P. Cohen, E. C. Miller, et al.
Cancer Res 1951;11:383-387.
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