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Dissociation and recombination of the subunits of human chorionic gonadotropin
Vol. 40, No. 2, 1970
BIOCHEMICAL
Dissociation
AND BIOPHYSICAL
and Recombination of the Human Chorionic Gonadotropin* N. Swaminathan Department University Buffalo,
State
ReceivedJune
RESEARCH COMMUNICATIONS
Subunits
of
and Om P. Bahl of Biochemistry of New York at Buffalo N. Y. 14214
11, 1970 SUMMARY
Human chorionic gonadotropin has been dissociated into two subunits by urea. They have been separated by chromatography on DEAB-Sephadex. The subunits are nonidentical as evidenced by electrophoresis, amino and carbohydrate composition and reassociate under suitable conditions. Leutenizing Ananthasamy, shown to
1967)
consist
chorionic
of
be composed
of
methyl
strongly
separated
dialysis. in
the
of one of
the
and isolated
(Canfield
erations
prompted
us to attempt
*Supported
from
A
simple
U.S.P.H.S.
the
422
bio-
on the
isolation
#lo273
prepara-
HCG, we
precipitated
the
during
to electro-
bands
1969).
for
also
The carboxamido-
subunits
--et al.,
Grant
their
of desialyzed
two broad
procedure
human
HCG might
HCG was subjected gel,
has been
Since
studies
two subunits.
served
HCG.
our
derivative
in
that
and 1969)
subunits.
During
polyacrylamide
of native
et - al -'
suggested
When carboxymethyl
phoresis
(Papkoff
(Reichert
two subunits.
derivative
ovine
(HCG) and LH resemble
of carboxamidomethyl
fortuitously
from
two nonidentical
it
behavior,
(LH)
and bovine
gonadotropin
logical
tion
hormone
were These of
the
separation
obconsidsubunits of
BIOCHEMICAL
Vol. 40, No. 2, 1970
subunits
of
HCG is
reported
The amino
acid
for
recombination
their
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in
the
and carbohydrate
present
communication.
composition
are also
and conditions
described.
EXPERIMENTAL AND RESULTS Separation two subunits of the
in
incubated to
column
of
0.04
developed designated fractions dialyzed giving
by 8 M urea. 1969)
HCG (Bahl, 0.04
at
diluted
with
was effected
purified
8 M urea
The dissociation
of Subunits.
sixty
250 ~1 with
with
a salt
against subunits
buffer
(0.9
gradient Fig.
each
and mixture
pre-equilibrated
pH 7.5.
The column
and two protein
peaks,
1, were obtained.
The
water
mg)
100 ~1 of
and transferred
peak were pooled,
distilled
in
x 30 cm)
buffer,
as A and B in comprising
above
HCG into (11.9
pH 7.5,
The incubation
A-50
M Tris-phosphate
buffer,
min.
the
DEAE-Sephadex
A sample
was dissolved
M Tris-phosphate
37O for
of
was to a
was
exhaustively
and lyophilized,
thus
A and B.
A
20
40 FRACTION
HCG. linear buffer,
60 No.
60
Fig. 1. DEAE-Sephadex chromatography of urea-treated The column (0.9 x 30 cm) was eluted with a continuous NaCl gradient (0.0 to 0.2 M) in 0.04 M Tris-phosphate Fractions pooled, pH 7.5. -----.
Vol. 40, No. 2, 1970
BIOCHEMICAL
Recombination and one minor band, gel
of IICG.
band whereas
when subjected (Fig.
2) in
When a mixture
sodium hours
subunit
37"
reassociated
B showed
to electrophoresis
2.5
in
buffer,
mg of
buffer,
each
pH
8.3
subunit
in
only
two major one broad
polyacrylamide (Davis,
4 ml
p H 6.8 was incubated
(Reichert
19691, -et -'al as evidenced by a single
electrophoresis
in polyacrylamide dissociation
that
the
could
also
be shown by polyacrylamide separation
Amino Acid The hydrolyses
1964).
of
0.02
M
carried
of HCG into
424
in
15-20
subunits
(Fig.
It
2).
subunits
may
prior
on DEAE-Sephadex.
5.7
of the
be
by urea
electrophoresis
Composition out
for
band on
by chromatography
and Carbohydrate were
the
gel
noted
to their
A showed
Disc electrophoresis of subunits A and B, HCG (A + B) and native HCG in polyacrylamide buffer, pH 8.3.
phosphate at
RESEARCH COMMUNICATIONS
The subunit
Tris-glycine of
Fig. 2. reconstituted gel in glycine
AND BIOPHYSICAL
Subunits.
N HCl in evacuated
Vol. 40, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Amino Acid Amino
Acid
Lysine Histidine Arginine Aspartic Threonine Serine
Values
tubes
acid
(Bahl
a Spinco
6.6 7.8 8.9 13.8 3.3 3.9 7.4 6.6 0.4 2.9
2.5 3.3 9.4 7.0 3.0 0.9 4.0 5.9 5.8
** Hydrolyses therefore Tryptophan
1.90 6.20 4.45 4.0
2.2
of the
polypeptide
content.
were carried out for 24 hours only and no correction was made for destruction. was not determined.
at llO"
for
and Smith, Automatic
24 hours.
19651,
the
Amino Acid
of A and B as seen
significant
differences. content
of
For
lysine,
methionine,
tyrosine
other
is
hand,
3.60 8.4 6.80 1.70
3.0
percent
5.15 2.40 9.93 8.46 7.20 7.26 9.79 11.16 2.90 3.69 8.28 6.88 2.06 2.32 6.93 4.28 3.61
10.80 10.35 2.90
8.4
composition
higher
5.65 2.50 7.45 8.50 7.30 7.50
1.1 10.7 9.7
7.3 8.0 7.2 12.7 6.9
represent
of KG HCG x
3.5
4.2
Acid
Subunit
B x
7.8 3.9
Acid Proline Glycine Alanine Half Cysteine Valine Plethionine Isoleucine Leucine Tyrosine Phenylalanine
sealed
of
A x
Glutamic
*
Composition*
rich
After
the
hydrolysate
Analyzer.
instance,
was analyzed
by
glutamic
A has much acid,
and subunit
prolinn,
acid
I reflects subunit
and phenylalanine in arginine,
the
The amino
in Table
histidine,
of
removal
B, on the
isoleucine,
and
leucine.
Neutral using
sugars
were determined
trimethylsilylether
described
(Bahl,
1969).
by gas chromatography
derivatives
as previously
Hexosamines
were determined
by an
Vol. 40, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE II Carbohydrate
Composition*
of
A
B
Sugar Galactose r4annose Fucose N-Acetylglucosamine N-Acetylgalactosamine Sialic Acid *
Uncorrected
Automatic
for
procedure
(1959).
recorded
considerably
than
in
of
HCG
4.51 5.10 0.73 7.95 1.09 7.00
4.30 4.30 0.60 7.10 1.70 7.20
of protein.
(Bahl,
1969)
and sialic
The carbohydrate Table
higher
HCG
A+B
7.50 4.80 1.30 7.40 2.00 10.20
content
Analyzer
monosaccharides
acid
water
Amino Acid
by Warren's
contains
1.52 5.40 0.36 8.55 0.19 3.90
Subunits
II
amounts
shows that of
acid
content
of
subunit
B
galactose
and sialic
A.
DISCUSSION
The results composed
presented
of two
dissociable
subunits.
by urea,
they
Although
previous
studies
terminal
analyses
suggested
identical
(Bahl,
electrophoretic acid
1969),
f the
composition .9
their tryptic subunits
nonidentity.
show clearly
that
Since
the
are
be attached
based
on amino
that
of the
were
remarkably
the
and carboxy
two subunits findings
and the
carbohydrate
subunits
definitely the
carboxamidomethyl different 426
HCG is
noncovalently.
present
Furthermore,
digest
subunits
must
the
mobilities,
above
might
be
on the and amino
establishes
finqerprints derivatives (unpublished
of the of results).
the
BIOCHEMICAL
Vol. 40, No. 2, 1970
Subunit showed
A showed
only
single in the
it
Since
Preliminary
results
acid
content
sialic
acid
might
for
studying
the
hormones.
It
subunit Since
polypeptide requisite
of the
subunits for
of
their
gonadotropic
of primary
therefore,
current
on the
elucidation
for
number
of subunits
is
an essential
structure,
the
of the
primary
or pre-
present
facilitate
greatly
HCG is
large
of other
separation
that
microheterogeneity.
structure
would,
the
this
method
investigation work
for
suggesting
a potential
study
of
The
offers
and their
the
for
content
differences.
of the
a
by preparative
to be suitable
a knowledge
chains
to give
acid
different
separation
found
preparation.
amino
however,
be responsible
and has been
scale
on the
B
of microheterogeneity
prexence
no significant
was,
The procedure
band whereas in
of A, obtained
showed
sialic
simple
the
components
electrophoresis,
and one minor
A and B reassociated
indicates
individual
RESEARCH COMMUNICATIONS
band on electrophoresis
gel.
band,
HCG.
two major
one broad
polyacrylamide
AND BIOPHYSICAL
the structure
of
hormone. REFERENCES
Bahl,
0. P.
Bahl,
0.
(1969)
P.,
240,
2. Biol. --
and Smith,
Chem.,
E. L.
244,
(1965)
3585.
567.
2. --Biol.
Chem.,
to be Canfield, R. E., Agosto, G. M. and Bell, J. J. printed in Proceedings of a Workshop meeting on Chemistry of Gonadotropins, held at University of Birmingham, England, September 1969. Davis,
B. J.
(1964)
Ann.
N_. Y_. Acad,
Papkoff, H. and Ananthasamy, T. S. Biophys. Acta, 147, 175. Reichert, L. E., and Midgley, Warren,
L.
(1959)
Rasco, M. A., A. R. (1969) 2. -Biol.
S&.,
121,
404.
(1967)
Biochim.
Ward, D. N, Niswender, 2. Biol. Chem., 244, -Chem., 234, 1971.
G. D. 5110.
BIOCHEMICAL
Dissociation
AND BIOPHYSICAL
and Recombination of the Human Chorionic Gonadotropin* N. Swaminathan Department University Buffalo,
State
ReceivedJune
RESEARCH COMMUNICATIONS
Subunits
of
and Om P. Bahl of Biochemistry of New York at Buffalo N. Y. 14214
11, 1970 SUMMARY
Human chorionic gonadotropin has been dissociated into two subunits by urea. They have been separated by chromatography on DEAB-Sephadex. The subunits are nonidentical as evidenced by electrophoresis, amino and carbohydrate composition and reassociate under suitable conditions. Leutenizing Ananthasamy, shown to
1967)
consist
chorionic
of
be composed
of
methyl
strongly
separated
dialysis. in
the
of one of
the
and isolated
(Canfield
erations
prompted
us to attempt
*Supported
from
A
simple
U.S.P.H.S.
the
422
bio-
on the
isolation
#lo273
prepara-
HCG, we
precipitated
the
during
to electro-
bands
1969).
for
also
The carboxamido-
subunits
--et al.,
Grant
their
of desialyzed
two broad
procedure
human
HCG might
HCG was subjected gel,
has been
Since
studies
two subunits.
served
HCG.
our
derivative
in
that
and 1969)
subunits.
During
polyacrylamide
of native
et - al -'
suggested
When carboxymethyl
phoresis
(Papkoff
(Reichert
two subunits.
derivative
ovine
(HCG) and LH resemble
of carboxamidomethyl
fortuitously
from
two nonidentical
it
behavior,
(LH)
and bovine
gonadotropin
logical
tion
hormone
were These of
the
separation
obconsidsubunits of
BIOCHEMICAL
Vol. 40, No. 2, 1970
subunits
of
HCG is
reported
The amino
acid
for
recombination
their
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in
the
and carbohydrate
present
communication.
composition
are also
and conditions
described.
EXPERIMENTAL AND RESULTS Separation two subunits of the
in
incubated to
column
of
0.04
developed designated fractions dialyzed giving
by 8 M urea. 1969)
HCG (Bahl, 0.04
at
diluted
with
was effected
purified
8 M urea
The dissociation
of Subunits.
sixty
250 ~1 with
with
a salt
against subunits
buffer
(0.9
gradient Fig.
each
and mixture
pre-equilibrated
pH 7.5.
The column
and two protein
peaks,
1, were obtained.
The
water
mg)
100 ~1 of
and transferred
peak were pooled,
distilled
in
x 30 cm)
buffer,
as A and B in comprising
above
HCG into (11.9
pH 7.5,
The incubation
A-50
M Tris-phosphate
buffer,
min.
the
DEAE-Sephadex
A sample
was dissolved
M Tris-phosphate
37O for
of
was to a
was
exhaustively
and lyophilized,
thus
A and B.
A
20
40 FRACTION
HCG. linear buffer,
60 No.
60
Fig. 1. DEAE-Sephadex chromatography of urea-treated The column (0.9 x 30 cm) was eluted with a continuous NaCl gradient (0.0 to 0.2 M) in 0.04 M Tris-phosphate Fractions pooled, pH 7.5. -----.
Vol. 40, No. 2, 1970
BIOCHEMICAL
Recombination and one minor band, gel
of IICG.
band whereas
when subjected (Fig.
2) in
When a mixture
sodium hours
subunit
37"
reassociated
B showed
to electrophoresis
2.5
in
buffer,
mg of
buffer,
each
pH
8.3
subunit
in
only
two major one broad
polyacrylamide (Davis,
4 ml
p H 6.8 was incubated
(Reichert
19691, -et -'al as evidenced by a single
electrophoresis
in polyacrylamide dissociation
that
the
could
also
be shown by polyacrylamide separation
Amino Acid The hydrolyses
1964).
of
0.02
M
carried
of HCG into
424
in
15-20
subunits
(Fig.
It
2).
subunits
may
prior
on DEAE-Sephadex.
5.7
of the
be
by urea
electrophoresis
Composition out
for
band on
by chromatography
and Carbohydrate were
the
gel
noted
to their
A showed
Disc electrophoresis of subunits A and B, HCG (A + B) and native HCG in polyacrylamide buffer, pH 8.3.
phosphate at
RESEARCH COMMUNICATIONS
The subunit
Tris-glycine of
Fig. 2. reconstituted gel in glycine
AND BIOPHYSICAL
Subunits.
N HCl in evacuated
Vol. 40, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Amino Acid Amino
Acid
Lysine Histidine Arginine Aspartic Threonine Serine
Values
tubes
acid
(Bahl
a Spinco
6.6 7.8 8.9 13.8 3.3 3.9 7.4 6.6 0.4 2.9
2.5 3.3 9.4 7.0 3.0 0.9 4.0 5.9 5.8
** Hydrolyses therefore Tryptophan
1.90 6.20 4.45 4.0
2.2
of the
polypeptide
content.
were carried out for 24 hours only and no correction was made for destruction. was not determined.
at llO"
for
and Smith, Automatic
24 hours.
19651,
the
Amino Acid
of A and B as seen
significant
differences. content
of
For
lysine,
methionine,
tyrosine
other
is
hand,
3.60 8.4 6.80 1.70
3.0
percent
5.15 2.40 9.93 8.46 7.20 7.26 9.79 11.16 2.90 3.69 8.28 6.88 2.06 2.32 6.93 4.28 3.61
10.80 10.35 2.90
8.4
composition
higher
5.65 2.50 7.45 8.50 7.30 7.50
1.1 10.7 9.7
7.3 8.0 7.2 12.7 6.9
represent
of KG HCG x
3.5
4.2
Acid
Subunit
B x
7.8 3.9
Acid Proline Glycine Alanine Half Cysteine Valine Plethionine Isoleucine Leucine Tyrosine Phenylalanine
sealed
of
A x
Glutamic
*
Composition*
rich
After
the
hydrolysate
Analyzer.
instance,
was analyzed
by
glutamic
A has much acid,
and subunit
prolinn,
acid
I reflects subunit
and phenylalanine in arginine,
the
The amino
in Table
histidine,
of
removal
B, on the
isoleucine,
and
leucine.
Neutral using
sugars
were determined
trimethylsilylether
described
(Bahl,
1969).
by gas chromatography
derivatives
as previously
Hexosamines
were determined
by an
Vol. 40, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE II Carbohydrate
Composition*
of
A
B
Sugar Galactose r4annose Fucose N-Acetylglucosamine N-Acetylgalactosamine Sialic Acid *
Uncorrected
Automatic
for
procedure
(1959).
recorded
considerably
than
in
of
HCG
4.51 5.10 0.73 7.95 1.09 7.00
4.30 4.30 0.60 7.10 1.70 7.20
of protein.
(Bahl,
1969)
and sialic
The carbohydrate Table
higher
HCG
A+B
7.50 4.80 1.30 7.40 2.00 10.20
content
Analyzer
monosaccharides
acid
water
Amino Acid
by Warren's
contains
1.52 5.40 0.36 8.55 0.19 3.90
Subunits
II
amounts
shows that of
acid
content
of
subunit
B
galactose
and sialic
A.
DISCUSSION
The results composed
presented
of two
dissociable
subunits.
by urea,
they
Although
previous
studies
terminal
analyses
suggested
identical
(Bahl,
electrophoretic acid
1969),
f the
composition .9
their tryptic subunits
nonidentity.
show clearly
that
Since
the
are
be attached
based
on amino
that
of the
were
remarkably
the
and carboxy
two subunits findings
and the
carbohydrate
subunits
definitely the
carboxamidomethyl different 426
HCG is
noncovalently.
present
Furthermore,
digest
subunits
must
the
mobilities,
above
might
be
on the and amino
establishes
finqerprints derivatives (unpublished
of the of results).
the
BIOCHEMICAL
Vol. 40, No. 2, 1970
Subunit showed
A showed
only
single in the
it
Since
Preliminary
results
acid
content
sialic
acid
might
for
studying
the
hormones.
It
subunit Since
polypeptide requisite
of the
subunits for
of
their
gonadotropic
of primary
therefore,
current
on the
elucidation
for
number
of subunits
is
an essential
structure,
the
of the
primary
or pre-
present
facilitate
greatly
HCG is
large
of other
separation
that
microheterogeneity.
structure
would,
the
this
method
investigation work
for
suggesting
a potential
study
of
The
offers
and their
the
for
content
differences.
of the
a
by preparative
to be suitable
a knowledge
chains
to give
acid
different
separation
found
preparation.
amino
however,
be responsible
and has been
scale
on the
B
of microheterogeneity
prexence
no significant
was,
The procedure
band whereas in
of A, obtained
showed
sialic
simple
the
components
electrophoresis,
and one minor
A and B reassociated
indicates
individual
RESEARCH COMMUNICATIONS
band on electrophoresis
gel.
band,
HCG.
two major
one broad
polyacrylamide
AND BIOPHYSICAL
the structure
of
hormone. REFERENCES
Bahl,
0. P.
Bahl,
0.
(1969)
P.,
240,
2. Biol. --
and Smith,
Chem.,
E. L.
244,
(1965)
3585.
567.
2. --Biol.
Chem.,
to be Canfield, R. E., Agosto, G. M. and Bell, J. J. printed in Proceedings of a Workshop meeting on Chemistry of Gonadotropins, held at University of Birmingham, England, September 1969. Davis,
B. J.
(1964)
Ann.
N_. Y_. Acad,
Papkoff, H. and Ananthasamy, T. S. Biophys. Acta, 147, 175. Reichert, L. E., and Midgley, Warren,
L.
(1959)
Rasco, M. A., A. R. (1969) 2. -Biol.
S&.,
121,
404.
(1967)
Biochim.
Ward, D. N, Niswender, 2. Biol. Chem., 244, -Chem., 234, 1971.
G. D. 5110.