Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PARTIAL AMINO ACID SEQUENCE OF RDMAN THYROXINE-BINDING GLOBULIN.
FURTHER EVIDENCE FOR A SINGLE POLYPEPTIDE Sheue-Yann
National
Received
Clinical of Arthritis, Bethesda,
Institute
November
CHAIN
Cheng
Endocrinology Branch Metabolism and Digestive Maryland 20014
Diseases
lo,1977
amino acid of highly purified thyroxineSummary : The NR2-terminal binding globulin has been identified by dansyl chloride, cyanate and All three gave alanine as the only amino Edman degradation methods. terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98-1.05 mole of alanine per mole of protein, These data further indicate that thyroxine-binding respectively. globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-ValThr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-~a-(Ser)-Leu-TyrA computer search revealed no homology of the NH2-terminal segment of The NR2-terminal thyroxine-binding globulin with human prealbumin. portion of prealbumin contains part of the thyroxine binding site. Thyroid globulin
hormones
(TBG),
three
the past
(1) *
Although
rations
from
molecular acid
decade,
a single
phoretic Nilsson amino
Abbreviation
composition, used:
(2),
that however,
the gel
are major
to 65,000
(1,2,3).
structure.
the sodium
suggested
and Peterson acid
subunit
characterized
Korcek
dodecyl
of the prepa-
discrepancies
(1,2,3),
Furthermore,
TBG consists
there
and Tabachnik
concluded filtration
of a single from their pattern
in
and in amino is
dis-
(4),
who
sulfate-polyacrylamide
TBG, human thyroxine-binding
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
the hormones
the homogeneity
there
36,500
composition
band in
pattern,
from
for
and partially
for
laboratories,
affinity
Among these
and 3,3'5-triiodo-L-thyronine.
was presented
ranging
the
and albumin.
TBG has been purified
and carbohydrate
found
prealbumin
serum L-thyroxine
different
about
in serum by thyroxine-binding
TBG has the highest
evidence
weight,
agreement
Copyright All rights
proteins, 75% of the
Over
transported
thyroxine-binding
transport
and carries
are
electropolypeptide
analysis
chain.
of the
in 6 M guanidinium globulin.
1212 ISSN
0006-291X
Vol. 79, No. 4, 1977
BIOCHEMICAL
chloride
and tryptic
composed
of four
Recently, polypeptide the enzymatic
which
MATERIALS
molecular
hydrolysis
(3)
presented
weight
filtration
analysis provide
on the subunit
acid
prealbumin,
evidence
of 54,000.
structure,
sequence
This
for
is
has not study
on
of TBG, on tryptic
of the NH2-terminal on the purity
a single
was based
in 6 M guanidinium
evidence
the quantitative amino
TBG, like
of the carboxyl-terminus
and on gel
not only
communication, the partial
--et al.
the quantitative
would
but also
with
that
subunits.
Gershengorn
peptide-mapping, However,
peptide-mapping
identical
chain
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chloride. amino
acid(s)
of the preparation
yet been reported;
of the NH2-terminal
In this residue
and
of TBG is described.
AND METHODS
Preparation of TBG. TBG was purified from 7 liters of pooled human plasma by a three-step purification procedure utilizing affinity, anion-exchange and gel filtration chromatography (3). The concentration of TBG was determined by its absorption at 280 nm, using EllZm = 6.2 (3). Potassium [14C]cyanate (51 Cifmol) was purchased from Amersham/ Searle. Dansyl chloride was obtained from Aldrich and recrystallized twice from hexane. Analysis of NH2-Terminal Amino Acid. Three methods were used to study the NH?-terminal residue of TBG: (a) Dansyl chloride method: Native TBG a;d TBG which was reduced and alkylated in the presence of 6 M guanidinium chloride (3) were treated with dansyl chloride and hydrolyzed as described by Gray (5). Dansylation of TBG was also carried out in the presence of 8 M urea (freshly prepared by ionexchange on mixed bed resins, AG501-X8). A solution of 0.8 mg of dansyl chloride in 40 ~1 of acetone was added to 0.5 mg of TBG in 0.1 ml of 0.2 M NaHC03, 8 M urea, pH 8.9 and kept at 25°C for 17 hours. Urea was removed by extensive dialysis against deionized water and the dansylated TBG was lyophilized followed by acid hydrolysis at 105°C for 24 hours. Two-dimensional thin-layer chromatography on polyamide sheets was used for the separation and identification of dansyl amino acids (6). (b) Cyanate method: Carbamylation was carried out in the presence of 6 M guanidinium chloride with potassium [14C]cyanate (157 mCi/mole) according to Stark (7). The NH2-terminal amino acid was quantified by amino acid analysis after hydrolysis of the hydantoin amino acid in 0.2 M NaOH at 110°C for 24 hours. (c) Manual Edman degradation: Three steps of Edman degradation was performed as described by Sauer --et al. (8). The phenylthiohydantoin amino acids were quantitatively identified by mass spectrometry (9), gas-liquid partition chromatography (10) and high speed liquid chromatography (11). NH2-Terminal Sequence-Determination by Automated-Sequencer. Ninetyfive nmoles of the fully reduced and alkylated TBG was subjected to automated Edman degradation in an updated Beckman-Spinco Model-890B Protein/ Peptide Sequencer using a Beckman program 111374. Phenylthiohydantoin amino acids were determined as described above and also by hydrolysis
1213
Vol. 79, No. 4, 1977
with (13)
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HI (12). Amino acid analysis using a Beckman 121 M analyzer.
was performed
as described
by Spa&man
RESULTS AND DISCUSSION Analysis with
of NH2-Terminal
dansyl
of dansilic was also
chloride,
dansyl
acid
found
were
carried
reduced
out
condition
gave alanine
as the only
reported
in Table
terminal
residue.
glycine
as the amino
glycine
per mole
When prealbumin,
both
prealbumin
alanine
obtained,
respectively.
hydantoin
alanine
However, for
the
carried tive these 1
two runs.
data,
under if it H.,
the absence
and 1.13
mole
of alanine
A lower
yield,
0.68
with
initial
Since
both
denaturation any,
is clear Lippoldt,
that
of manual yields
conditions,
R.E.,
one mole
and Robbins,
1214
Edman (see Table
were
obtained
and Edman degradation the possibility
J.,
for
be very
was liberated personal
with
of phenylthio-
Edman degradation
of alanine
amino
obtained
from automated
should
of
of TBG was
mole
and 0.98
residue
carbamyl
per mole
and 0.82
with
a recovery
or the recovery
of 1.02
carbamylation
reported
of other
recovered
due to unaccessibility that
(7)
0.76
per
of
protein
similarly,
(0.98)
amino
of alanine
characterized
factor
the extrapolated
subunit,
in
only
are
and hydrolysis
Stark
per mole of TBG, were
as compared
out
Edelhoch,
was obtained.
correction 0.88
a well
Both
by carbamyl-
as the residue
isolation
was carbamylated
and glycine
Stark's
(0.76),
degradation
terminus
of protein
gave alanine
made for
The
The results
of TBG gave 0.86
no correction
was
backbonel.
was obtained
Edman degradation. methods
of TBG
chloride.
the polypeptide
acid
three
Dansylation
of 6 M guanidinium
amino
All
amount
and on TBG which
of the NH2-terminal
hydantoin.
Using
of 8 M urea
acid.
I.
and trace
hydrolysate.
amino
and automated
TBG was treated
lysine
NH2-terminal
alanine
acids.
in the acid
Carbamylation
of TBG with
of 98% for
NE-dansyl
in the presence
and by manual
mole
When native
has been shown to unfold
Quantitation ation
alanine,
in the presence
and alkylated
latter
Amino Acid.
were an unreac-
remote.
From
per mole
communication.
I).
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table NB2-Terminal
Amino Acid
Identification Method Dansyl
chloride
Manual Edman Degradation
Alanine
1.05c,
Automated Edman Degradation
Alanine
0.68 0.82
weight
of 54,000
by amino isolation
from
was used for
TBG
1.04d (1.02), (0.98)e
(3).
acid analysis and not corrected for and hydrolysis of hydantoin alanine.
c,d Determined as phenylthiohydantoin alanine liquid partition and liquid chromatography,
by gasrespectively.
eValues are from two separate runs and were by liquid chromatography. The corresponding initial yields are in parentheses.
determined extrapolated
This
evidence
is consistent
of a single
Automated
and serine-18,
with
polypeptide
Edman Degradation
of TBG are
of TBG.
shown in Table
of protein.
were
II.
(68 and 82%),
The results
of the
sequence
For all
yield
calculated run
is
extrapolated
further
residues
at step using from
1 and run
of the TBG purified
The purity and the
(3,4)
residue
1 and 9 was 92% and 90% for
(3).
reports
made in two experiments
The repetitive
The homogeneity
the earlier
that
chain.
and one unidentified
uous assignments
shown
TBGa
Alanine 0.86b
TBG consists
been
Mole/mole
Alanine
losses
in step
Globulin
Cyanate
b Determined
analysis
of Human Thyroxine-Binding
NB2-Terminal Amino Acid
aMolecular
of TBG.
I
16,
except identical
different
initial
1215
yield
unambigpreparations
the recovery
of alanine
2, respectively.
by the three-step demonstrated
proline-
by the
procedure initial
(102 and 98%).
had yield
Moreover,
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Automated
Amino
Terminal
II Degradation
Thyroxine-Binding
Step
Amino Acidb
1
Alanine
2
Serine
3
Proline
Mole/Mole
TBG
0.82
of Human
Globulina
Identification GC LC
MS
f
+
-I-
+
+ +
+
+
+
+
0.57
+
6
Lysine
0.73
+
+ + +
7
Valine
0.53
+
+
a
Threonine
4
Glutamic
Acid
5
Glycine
0.58
+
+ +
+ +
+
9
Alanine Aspartic
11
Serine
+
+
12
Serine
+
+
13
Serine
+
+
14
Glutamine
+
+ +
+ +
f
+
+ + +
15
+
0.25
+
10
Acid
0.37
MethodC AAA
(Proline)
+ +
16
X
17
Alanine
ia
(Serine)
19
Leucine
0.07
+
+
20
Tyrosine
0.08
+
+
0.10
+
+ +
aThe data presented are from one of two experiments which gave virtually identical results. The starting TBG was 95 nmoles. The yields were determined as phenylthiohydantoin amino acids Only the quantifiable phenylthioby liquid chromatography. hydantoin amino acids were estimated. b The parentheses and unidentified
and symbol residues,
x indicate respectively.
tentative
assignments
'The identification methods are: GC, gas-liquid partition chromatography; LC, high speed liquid chromatography; MS, mass spectrotrometry; and AAA, amino acid analyses of HI hydrolysates.
1216
Vol. 79, No. 4, 1977
there the
BIOCHEMICAL
is no other first
identifiable
ecule.
The TBG used for
batches
of pooled
of variable preclude
possible
Furthermore,
these
NH2-terminal
acid
present
study
to prealbumin,
based
in
binding
tallography
(17).
same biological
is conceivable
site
function
that
similarity
in the
thyroxine
transport
earlier of a single
using
identity
of the and that
labeling either tertiary
labeling
fact
that
lysines
of TBG with
does not
it
is
clear
polypeptide
and a
no homology
of the
sequences as part
and X-ray
two proteins
identified
that
chain.
matrix
was found (15,16)
these were
The absence
1050 protein
of prealbumin
by affinity
(3),
revealed
probabilities,
Lysine-15 both
two 7-liter
in the molecule.
scoring
of TBG to any of the current
In view
by affinity
consists
on mutation
from
of the mol-
obviously
elsewhere
presented
searches
prealbumin.
the hormone
sequence
expressed
and evidence
portion
from 28 individuals.
in the NE2-terminal
segment
including
was purified
each derived
two computer
matrix
studies
gene mutations
TBG, in contrast
residues
amino
in the NE2 -terminal
sequence
plasma,
residues
From this
(14)
phenylthiohydantoin
step.
TBG shows a unique
scoring
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of crys-
have the
as the binding
N-bromoacetyl-L-thyroxine2,
homology
in sequence
structure,
or both,
in the binding may exist
between
it domain these
or two
proteins.
ACKNOWLEDGEMENTS The author wishes to thank Drs. Ettore Apella valuable help with the automated sequence analysis sions, and Dr. Jacob Robbins for critical reading is also grateful to Ms. Elisabeth Robinson for the hydantoin amino acids by liquid chromatography and the amino acid analyses.
and Thomas Fairwell for and stimulating discusof the manuscript. She analysis of phenylthioMr. Jonathan Seeman for
REFERENCES 1. 2.
2
Robbins, Clinical Alan R. Nilsson, 8543-8553.
Cheng,
S.-Y.
J. (1976), in Trace Components of Plasma. Isolation and Significance, (G.A. Jamieson and T.J. Greenwalt, eds.) Liss, New York, pp. 331-350. S.F., and Peterson, P.A. (1975), J. Biol. Chem. 2,
and Robbins,
J.,
unpublished
1217
results.
Vol. 79, No. 4, 1977
3. 4. 5. 6. 7. a. 9.
10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Gershengorn, M.C., Cheng, S.-Y., Lippoldt, R.E., Lord, R.S., and Robbins, J. (1977), J. Biol. Chem. 252, in press. Korcek, L., and Tabachnick, M. (1974), Biochim. Biophys. Acta 371, 323-336. Gray, W.R. (1972), Methods Enzymol. 5, 121-138. Woods, K.R., and Wang, K.T. (1967), Biochim. Biophys. Acta 133, 369-370. Stark, G.R. (1972), Methods Enzymol. 2, 103-120. Sauer, R.T., Niall, H.D., Hogan, M.L., Keutmann, H.T., O'Riordan, J.L.H., and Potts, J.T., Jr. (1974), Biochemistry 13, 1994-1999. Fairwell, T., and Brewer, H.B., Jr. (1973), Fed. Proc. 32, 648. T.J. (1969), J. Biol. Chem. 244, Pisano, J.J., and Bronzert, 5597-5607. Zimmerman, C.L., Apella, E., and Pisano, J.J. (1976), Anal. Biochem. 75-, 77-85. Smithies, O., Gibson, D., Fanning, E.M., Goodfliesh, R.M., Gilman, J.G., and Ballantyne, D.L. (1971), Biochemistry lo, 4912-4921. N.M. (1958), Anal. Chem. Spackman, D.H., Moore, S., and Stein, 2, 1190-1206. Dayhoff, M.O. Hunt, L.T., Baker, W.C., and Schwartz, R.M. (October, 1977), Protein Sequence Data File, National Biomedical Research Foundation, Washington, D.C. Cheng, S.-Y., Cahnmann, H.J., Wilchek, M., and Ferguson, R.N. (1975), Biochemistry 14, 4132-4136. Cheng, S.-Y., Wilchek, M., Cahnmann, H.J., and Robbins, J. (1977), J. Biol. Chem. 252, 6076-6081. S.J. (1977), Nature 268, 115-120. Blake, C.C.F., and Oatley,
1218