The amino acid sequence of a 27-residue peptide feleased from the α-chain carboxy-terminus during the plasmic digestion of human fibrinogen

BIOCHEMICAL

Vol. 71, No. 3,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE AMINO ACID SEQUENCE OF A 27-RESIDUE CARBOXY-TERMINUS

DURING THE PLASMIC DIGESTION OF HUMAN FIBRINOGEN

Barbara Department

A. Cottrell

of Chemistry,

and Russell University

La Jolla,

Received

PEPTIDE RELEASED FROM THE a-CHAIN

F. Doolittle of California,

California

San Diego

92093

June 7,1976

SUMMARY: The amino acid sequence of a 27-residue peptide released during the early stages of the plasmin digestion of human fibrinogen has been determined, The corresponding cyanogen bromide fragment has also been isolated from the purified a-chains of fibrinogen, although a separable fraction of those chains lack the fragment, evidently because of in vivo degradation. The peptide is the carboxy-terminal segment of nativechains. During

the

course

of characterizing

of the human fibrinogen tain

homoserine

peptide. ready

and which

We were determined

fragments

nized

a-chain

a-chain

unusually

for

high

the appropriate

histidine plasmic

plasmin-generated

peptide

bromide

which

fragment,

of human fibrinogen (l-3).

of considerable

with

cyanogen content peptide is

of the

only

is to say it

attempt. acid

longer

has an amino-terminal

residue.

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

we were

754

them to

the

carboxy-

of its

recog-

proteases

(4).

to plasmin

similar

fragment.

peptide,

one amino

bromide

exposure

properties

on our first

cyanogen

because

and other

bromide

not con-

we had al-

and found

interest

to a brief

a peptide

did

since

Beyond that,

by plasmin

fibrinogen

carboxy-terminal

peptide

of the carboxy-terminal

to proteolysis

specifically

which

fragments

to the carboxy-terminal

in this

segments

is

we subjected

searched the

homologous

vulnerability

Accordingly,

sequences

bromide

a peptide

corresponded

interested

B- and y-chains

have structurally of the

we isolated

therefore

especially the

of the

terminus

a-chain,

the cyanogen

to those Because able

of

of the

to identify

Fortuitously, than

and

the

the cyanogen

methionine

Vol. 71, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL

EXPERIMENTAL Preparation pared

according

alkylated

(Fig.

dialyzed which

chromatographed

exhaustively

against

subjected

bromide

peptides

purified

was observed

that

the

represented

the

2).

of the

dissolved

was added

10 min.

at room temperature, soybean

the reaction served

0.4 mls

a Sephadex

for the

peptides

by centrifugation

were

(2.5

The small

0.1 M ammonium bicarbonate. were

subjected

containing mobility

to paper

peptides as the

with

cyanogen

Pauly

bromide

1 A complete report dealing with will appear elsewhere (7).

the

reagent

755

supernatant

strips

of

as by placing was ob-

parent

pro-

was passed

over

and developed

described

the characterization

After

the precipitated

(6).

M Tris,

3).

and medium molecular and the

fragment

(Fig.

equilibrated

electrophoresis the

of digestion

from

0.05

by the addition

The extent

min);

x 100 ems.)

M NaCl,

as well

separated

x g,lO

To approximately

(Worthington),

on SDS gels

and thus

50% glycerol.

was halted

10 min. digests

(20,000

G-50 column

digestion

inhibitor

at 65'

by electrophoresing The released

tein

the

in

I).

of 0.15 in

It

was absent

homoserine

(Table

(Kabi)

weight

at pH 2 and 5.

Fragment.

20 mls

of human plasmin

trypsin

mixture

in

The

x 90 ems) equilibrated

contain

chains

Plasmic

(6).

in 10% acetic

a band which

not

of those

following

and medium molecular

exhibited

Corresponding

pH 7.5,

(2.5

electrophoresis

did

separately,

bromide

dissolved

column

and

cellulose

were'pooled

cyanogen

The small

The peptide

220 mg of human fibrinogen

4 mg of solid

1

a2 pools

carboxy-terminus

Isolation

G-50

by paper

one of the

the case of u1 (Fig.

with

pre-

was reduced

and lyophilized,

and then

same solvent. further

peaks

water,

on a Sephadex

with

were

distilled

(5)

on a carboxymethyl

a-chain

was lyophilized

and fractionated

and developed

procedure

to fragmentation

digest

Human fibrinogen

Fragment.

preparation

Two distinguishable

were

Bromide described

1).

they

cyanogen

Cyanogen

to a previously

and the

column

acid

of the

weight

scanned

A peptide

with

with

pools

for

histidine-

the

same

above was isolated

of each of these

pools

Vol. 71, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FRACTION

Fig.

1

NUMBER

Carboxymethyl cellulose chromatography (2.5 x 20 ems) of human fibrinogen wiiich has been reduced Cdithiothreitol) and alkylated (14-C iodoacetic acid). The starting buffer is 0.01 H sodium acetate, pH 5.2, in 8 M urea. The elution gradient was achieved by mixing equal volumes c400 mls each) of starting buffer and a limit buffer composed of 0.15 M sodium acetate, pH 5.2, in 8 M urea.

0 !: : N +

-

+

Fig.

2

and found

Paper electrophoretic guidestrips a peptide in one of the cyanogen is absent from the corresponding phoresis conditions: pH 2.0, 300 to be the corresponding

position

is

identical

presence

of one additional

shown to be at the

with

plasmic

the cyanogen residue,

amino-terminus

stained with ninhydrin showing bromide pools of a2 chains which pool of al chains. ElectroY,' 3 hrs. fragment.

Its

bromide

peptide

a methionine

which

(Table

756

I).

amino except

acid for

comthe

was subsequently

BIOCHEMICAL

Vol. 71, No. 3, 1976

TABLE I.

Amino

Acid

Fragments

Compositions

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Carboxy-Terminal

from Human Fibrinogen

Cyanogen

Bromide

and Plasmin

a-Chainsa

CNBr Peptide

Plasmin

Peptide

HaCN5c

HFPL3A2

2.1

(2)

2.2

(2)

Threonine

1.7

(2)

1.6

(2)

Serine

3.1

(3)

3.1

(3)

3.2

(3)

3.2

(3)

Proline

1.0

(1)

0.8

(1)

Glycine

3.3

(3)

3.2

(3)

Alanine

4.1

(4)

4.0

(4)

Valine

0.9

(1)

0.9

(1)

0.8

(1)

Aspartic

acid

Glutamic

acid

Methionine Histidine

2.8

(3)

3.0

(3)

Lysine

2.1

(2)

2.8

(2)

Arginine

2.0

(2)

1.8

(2)

Total

Residues

26

aExpressed as residues per mole. leucine, tyrosine, phenylalanine, on a Spinco Model 119 Amino Acid column system. Amino Acid two peptides

determined

(9).

Preparations

using

phenylene

the (12).

largest

Determination.

were

attached

main

The amino

bromide

an automatic

solid

and plasmic phase

(10)

stepwise

by a thioacetylation

regard,

subpeptides the

subpeptides

tryptic

peptide

cyanogen characterized also

757

of our beads

fragment I).

to the

were,

in the

own design (Corning)

(11).

A thermolysin peptide

(111,

In addition,

DNS-PITC

was digested

a key confirmatory

of the

carbodiimide

procedure

(Table

yielded

soluble

subjected

bromide

sequences

fragments)

glass

or a water

were

acid

sequencer

to aminoalkylated

diisothiocyanate

and certain

In this

and three the

with

degraded

peptides

Neither of the peptides contains isoleucine, tryptophan or cysteine. All results obtained Analyzer employing a three-buffer, single

the cyanogen

(i.e.,

main,

and then

Sequence

27

procedure with digest

trypsin of

(Thr-Lye.).

BIOCHEMICAL

vol. 71, No. 3,1976

Fig.

3

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Dodecyl sulfate (SDS) gel electrophoresis of fibrinogen Electrophoresis and after minimum digestion with plasmin. 4% acrylamide gels, 9 mAmps-/gel, pH 7.2 (14).

before conditions:

DISCUSSION The complete in Fig.

4, along

amino with

posed terminal

as the y-chain

of the

carboxy-terminal

cyanogen

of 26 and 27 residues valine,

sequence

The a-chain

of human fibrinogen. sane size

the

acid

an observation

plasmin-derived

sequences

cyanogen bromide

respectively. consistent

758

bromide

peptide

of the fragment

fragment,

both

is

with

a report

peptides that

shown

B- and y-chains

the peptides

Also,

is

almost being

the con-

have carboxythe carboxy-

Vol.71,No.3,1976

BIOCHEMICAL

CARBOXY-TERNIKAL

AND BIOPHYSICAL

CYANOGEN BROMIDE FRAGMENTS OF THE INDIVIWAL

RESEARCH COMMUNICATIONS

CHAINS

FROn HIJWN FIBRIWGEN

Plasmin CN ..MET~A~~P-GLU-AUI~LY-SER~L"-ALA-ASP-~IS-GL"-GLY-THR-HIS-SER-THR-LYS-ARG-GtY-"IS-A~-LYS~~-~~-PR~V~c

p

F

..MET-LYS-ILE-AR&PRO-PHE-PHE-PRO-GLN-GLN' CN t t ..MET-LYS-ILE-ILE-PRPPHE-ASW-ARt-LEU-THR-ILE-GLY~LU~LY~LN~LN-HIS-HIS-L~~LY~Y-AU-LYS~N-~~Y-~P-V~c

Y

t - Crosslinking

Fig.

4

residues

of the

features,

any further

to the

strong

are both

resemblance homology

Acceptor

Site

segment of human fibrinpeptides from B-chains

valine

(13).

Except

must be regarded

existing

between

for

as marginal

'& and y-chains

4). It

is generally

a-chains

are

as

Mills

1970,

ogen were being

very

weight.

indistinguishable

with

plasmin

calculated the

responding reported

(4).

is

from

the

its

that

differing of these

smaller

constituent

previously. that

reported Although

the

we would

feel

3000 in

could

be converted treatment

amino

acids

weight is

of two distinct

SDS gel

and Karpatkin system

comfortable

759

of human fibrin-

by approximately

molecular

by Mills

ago

there

the

and characterization

As long

electrophoresis,

by short-term

that

of fibrinogen

attack.

the a-chains

by SDS gel

larger

noteworthy

halves

to proteolytic

species the

from It

carboxy-terminal

reported

when examined

to the bands

resolving

(4)

Moreover,

isolation

the

and vulnerable

two predominant

form

know,

exposed

that

and Karpatkin

at least

peptide

accepted

heterogeneous

molecular

highly

) = Crosslinking

a- and y-chains

however,

in contrast

at best, (Fig.

Site

Amino acid sequence of the carboxy-terminal ogen a-chains compared with the corresponding (3) and y-chains (1,2).

terminal these

Donor

t

of fibrinogen

So far

a-chain

as we

types

car-

has not been

we use (14) attaching

a

of our plasmic

2,859.

(4)

into

is

a value

not

so

to the

Vol. 71, No. 3, 1976

small

size

that

BIOCHEMICAL

difference

between

the difference

residue

carboxy-terminal In recent

molecular that

between

being

This

A (16). inantly

moiety

a part

corresponding

been reported

equivalent

fragment

plasma

contains

27-residue

activity fibrin

both

large

domains

is

little

doubt

of the plasmin-released

27-

a1 -chains that

weight will

this

be a very before

Similarly,

peptide

suggesting

employing

of these

unusual

if

the

freshly

collected

that

removal

of this

--in vivo.

day-by-day

degradation directed

as probes polar

to find

appropriately

way to monitor

may be useful

by plasmin

from

circumstances

antibodies

fragment

homoserine.

prepared

more extensive

or fragment

released

be of interest

ordinary

simple

bromide

material

is missing

peptide

H (15)

composition, being predom-

acid

A cyanogen

fibrinogen

under

of fibrinogen,

to as fragment

acids.

and it

on a much larger

plasminolysis

amino

a1 and a2 chains,

for

of influence

the

molecular

C17),

has occurred. of this

there

has been placed

referred

amino

occurs

at a stage

terminus

during

be noted

a radioimmuno-assay might

released

from

peptide

antibodies

emphasis

has a remarkable

to this

should

the absence

a major

of polar

has also

It

them is

of the a-chain

material

composed

our a 1 and a 2-chains,

peptide.

reports

weight

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

As such,

directed plasmin

of fibrinogen to the

and/or

carboxy-

characterizing

the

extremeties.

ACKNOWLEDGMENTS This

study

was supported

by N.I.H.

Grants

No. HE 12,759

and GM 17,702.

REFERENCES 1.

2. 3. 4. 5.

Chen, R. and Sharp, J.J., 25:334-336 TaEgi, T. and Doolittle, R.F. Biochim. Biophys. Acta, 386:617622 (19752. Mills, D. and Karpatkin, S. Biochem. Biophys: Res; Comm., -40:206211 cl9701. Doolittle, R.P., Schubert, D. and Schwartz, S.A. Arch. Biochem. Biophys., 118:456-467 09671.

760

Vol. 71, No. 3,1976

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Gross, E.'Metlicds Enzjmcl., 11: 238-255 (1967). Doolittle, R.F., Cassman,K.G., Cottrell, B.A., Friesner, S., Hucko, J and Takagi, T. In preparation. Smi;;, I. in Chromatograpliic and Electrophcretic Techniques, Vol. 1, p. 296. Interscience: New York, 1960. L. and Doolittle, R.F. Doolittle, L.R., Mross, G.A., Fothergill, In preparation. Laursen, R.A., Horn, N.J. and Bonner, A.G. FEBSLetters, 21:67-70 (1972). Mross, G.A. and Doolittle, R.F. Fed. Proc., 30:1241 abs. (1971). Gray, W.R. Methods Enzymol., 25:333-344 <19??!). Okude, M. and Iwanaga, S. Bioxim. Biophys. Acta, 251:185-196 (1971). Weber, K. and Osborn, M. J. Biol. Chem., 244:4406-m2 (1969). Harfenist, E.J. and Canfield, R.E. BiocheGtry, 14:4110-4117 (1975). Takagi, T. and Doolittle, R.F. Biochemistry, 14:5=9-5156 (1975). Gollwitzer, R., Becker, U., Hahn, E. and TimplTR. FEBSLetters, 36:281-284 (1973). -

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