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