Fragments resulting from CNBr cleavage of bovine fibrinogen and its D and E derivatives

THROMBOSIS Printed

RESEARCH in the United

States

RESULTING

FIBKINOGFN

M K loczewiak.

FROM

AND ITS

of Radiobiology

at the International tives,

(Received

OF ROVINE

A Pankiewicz.

Z. S. Latallo

M .K opeC Protection

Warsaw,

October

8.12.1972; Accepted by

Institute

of Nuclear

Poland

Conference

Warsaw,

CLEAVAGE

AND E DERIVATIVES

and Health

Research, Presetned

D

CNRr

G . Dudek- Wojciechowska, and

Department

271-282, 1973 l’ross, Inc.

COMMUNICATION

BRIEF

FRAGMENTS

vo1.2, PP~ Pereamon

on. Fibrinogen

24 -

and its deriva-

26, 1972.

in revised form 8.2.1973. Editor B. Blombgck)

INTRODUCTION ________________

Our previous molecular tive

studies

weight

proteolysis

present

fragments of bovine

in fibrinogen

that time we have knot

( N-DSK)

disulphide

bridges

our data provided each other.

two fragments overlapping

fibrinogen

molecule

described

cule of fibrinogen

(fragments

postulated

Immunological

overlap

on the amino acid composition

are

revealed recovered

that apart

by Blomback

( “disulphide studies very

D and E)

strong

Since

in these

et al.

products

/2/ other

D the existence

of a pair

proteolysis

from

disulphide rich

in

in fibrinogen

/3/.

and fragment

as E

/5/ that one moleone fragment

E and

“disulphide

knots”

appeared

to be an

of symmetrical

D and different

/ 11. At

at the same meeting

that the N-DSK

upon plasmin

an exhaus-

fragments

might be present

/4/ presented

evidence

from

that all the half-cystines

it has been shown previously

produces

with fragments

resulting

from the N-terminal

knots”)

on N-DSK

of the two main high

N -DSK

272

DISULPHIDE

obvious

possibility

In order

subjected

this possibility

been isolated

to CNBr

product

resulting

G-100

chromatcgraphy

bovine

/l,

6 S-S

the original These

data are

et al.

/lo/

E CNBrI.

indicated pair

that besides

in fibrinogen

molecule

Therefore, from

the disulphide

studies

fibrinogen

fragment

large

molecule

on separation were

/9/ reveaE from

only

4 out of

work

strongly

occur

knot”

although discussion).

the hypothesis

in fibrinogen

with fragment

in disulphide located

of disulphide

(see

supported

fragments

and

E a

bridges

in fragments

rich

D /6/.

of Gardlund

human fibrinogen

differences

partly

DCNBrI

of fragment

of the recent

rich

for

contained

knot overlapping

at least

in the ultracen-

of the fragment

of “disulphide

regions

at pH 3.5,

content

molecule

D from

corres-

to be homoge-

34 700 Daltons

DCNBrI

here

than one type

estimated

bridges

of fragment

and are

were

7 M urea

for the cystine

due to species

fairly

of symmetrical,

/8/ were

summarized

of more

by Sephadex

products

size

with the results

probably

Our own data briefly of the existence

was isolated

found in the parent

cleavage

purification

( 7.5%,

Assay

3/ whereas

comparable

and after

the main high molecular

These

all 9 disulphide

bridges

on CNBr

some differences

acid.

molecular

sedimentation

I contained

/6/ fragments

and ECNBr I. Both appeared

. Their

for

fibrinogen

cleavage

gel electrophoresis

by equilibrium

led that ECNBr

fibrinogen

In each case

CNBr

as DCNBrI

buffer)

and 33 000 Daltons

bovine

/7/.

from

nous in polyacrylamide

trifuge

vo1.2,No.3

studies

in our further

in 10% acetic

designated

0.075 M formate

from

treatment

weight

pondingly

FRAGMENTS

.

tc prove

D and E have

RICH

exist D. directly

performed.

RESULTS -____-------Purified

bovine

fibrinogen

in 70% formic acid according /7/.

A mixture

.polyacrylamide

of products

(fraction

I - 4)

to the procedure resulting

gel electrophoresis

/ ll/

was treated

described

from this treatment ( 7.5%,

7 M urea

with CNBr

by Cahnman el al. was analyzed

at pH 3.5,

0.075

by M

DISULPHIDE

Vo1.2,No.Y

formatc

hfrcr)

3) . Some, tained

Iwo

attempts

designated FCNBrII

bands

but not a clear

by Sephadex

rious

main

prc-vailing

moving)

mobilities

relatively

A mixture

exhaustively protein

dialyzed

againsl

fragments

ammonium

sulphate

10% acetic

acid

cipitation Two

steps

saturation.

and dialyzed

Separation

tes the fraction

CNBr)

concentration. lumn equilibrated

and

elaborated.

of fibrinogen

acid.

0.5%

prccipitotc

the

mobility I)rought

precipitate

10% acetic

containitlg

saturation,

electrophoretic

was

(condi-

up to 0.25

was dissolved

All

dialysis

in

and pre-

containing

l-2% of protein

G-100

( 1.9x110

was subjeccm)

cquilit)ro-

acid.

pattern

out at room temperature is shown on I;ig.

containing

material

was freeze

2SO4

on Sephadex

fragment

by rechromatography

starting

cleavage

.l’l~c! solution

The resulting

of this

- dried

(dialyzed

under

of I2 ml per arca

indicn-

purification was . the same conditions.

FCNBrIl

products

solution

with 10% acetic

Further

fraction

and dissolved

One ml of this

at a rate

1. ‘I‘hc shadowed

F CN BrI.

Isolation and purification of fragment _____________________~~______~~______~~~~~

The

tentatively

( slou er)

were

and tllc supcrnatnnt

solution

was carried

The elution

FCNBrI

va-

out at +

chromatcgraphy

ted urith 10% acetic

CNAr

and higher

against

carried

After

fragments

procedures

waler.

(NII4)

discarded

ml of the dialyzed

ted to column

achieved

was

were

from

at 0.025

acid.

was oh-

FC_BrI

dislilcd

of lower

3)

as on Fig.

hour.

resulting

was prccipitotcd

containing tions

of products

fragment

l’ig.

( see

main products

of these

simple

Isolation and purification of fragment ____________-_______~~~~~~~~~~~~~~~~~~~-~

pnt(cni

it) loo/, acetic

and purification

to their

273

iI1 tllc

of these

chromatography

for isolation

(faster

wcrc

FRAGMENTS

cut separation

G-100

according

RICH

acid

of fibrinogen

in lQ% acetic

was applied ( 1.1x93

cleavage

by

acid to l-2% protein

on Sephadex

G-100

cm) . ‘1‘1IC scpardtian

cowas

274

DISULPHIDE

RICH

FRAGMENTS

l.0 0.9

as 02cm

07

E280nm

t

06

0.5 -

0.5

0.4 -

0.4

03-

0.3 0.2

0.2-

0.1

0.1-

0.0 0 40 80 120 160200240 280d Fig. Sephadex

0

1

Fig.

Sephadex

G- 100 chromatography

carried

out at room

pattern

obtained first

temperature

is presen

peak

than fragment

F CNBrI

lity

II.

as

previous The

FCNBr

separation second

acetic sulphate

acid

was

In the following

to 1% protein

saturation,

collected

CT 8 ml per hour.

FCNRrI,

material

The

elution

several

of the same

in column

size

fragments clectrophoretic between

this

slower mobiand

. as

shown

step

concentration

at + ho.

G- 100 chromatcgraphy

2.

the difference

of FCNBrI)

peak

and freeze-dried.

fragment

and some

(Note

at a rate

ted on Fig.

contained

2

of the dialyzed products of CN Br cleaved fibrinogen. Shadowed area indicate fraction used for further purification of fragment FcNB~II by ammonium sulphate precipitation. Column 1.1x93 cm.

of fraction precipitated at 0.25 ammonium sulphatc saturation from the products of CNBr cleaved fibrinogen. Shadowed area - FCNBrI. Column 1.9x110 cm.

The

74 28 52 56 70 80 92ml

this

by the

shadowed

fraction

and precipitated

was

area

dissolved at 0.1

on Fig. in 1@:6

ammonium

2

DISULPHIDE

Vo1.2,No.3

Fig. 3 Polyacrylamide

275

RICH FRAGMENTS

b

a

c

d

e

f.

gel elec

trophorcsis of products of CNBr treated fibrinoden (a)

; fragment

FCNRrI treated

(1,) ; FCNRrI with thrombin

(c) ; ECNBrI (see text) (d) ; FCNnrIl (e) and

.(7.5% gel, urea, 0.075 M for-

DCNBrI(f)

7M

mate buffer pll 3.5, staining with amido black) .

‘l‘he precipitate

contained

The two fragments as judged

Preliminary

for

FCNBrI

are necessary Treatment sulted

for

effect

precise

change

presenting man N-DSK.

molecular

size

based

sepa-

on sedimenta-

More

ultracentrifuge

37O, pH = 7.8 for

in electrophoretic in case

mobility of F CNRrII

in molecular ( Table

of h uman fibrinogen

runs

of bovine

F

re-

CN BrI

3) .

(Fig.

to identify

elution

disulphide fragment

fibrinogen.

moving

as observed

The amount of these

of fragment

1) , to the N-terminal

/ 2, lO/ allow

by slower

N-DSK

120 min)

size, chromatogrnphic

FCN Br II was quite homogenous

oligomeric

well

of about 60 000 and 40 000

values

(10 NIH u/ml,

content

F CN RrI was contaminated

to be very

estimation.

and similarities

1;cN BrI as the N-DSK Fragment

fragment

3) .

respectively.

was observed

and half-cystine

knot (N-DSK)

of their

and FCNBrII

no change

‘[‘his

(Fig.

/8/ inidicated

with thrombin

in a drastic

whereas

pattern

by electrophoresis

assay

II appeared

and FCNBr

FCNBrI

measurements

tion equilibrium Daltons

homogenous

3) .

F cN 13,lI ( Fig.

rated

electrophorctically

heavier

in electrophoresis components, by Rlomback

components

whereas

most likely et al.

re-

/12/ in hu-

apparently

increased

DISIJLPHIDI? RICH FRAGMENTS

276

TABLE Comparison

Vo1.2,No.Y

1

of the data concerning

half-cystine

gen and its various

content

in bovine

fibrino-

derivatives

~~~~~~~~-~~--~~~1-------------~~~--------~~~~~~~~~~~~~~~~~______~_ Molecular llalf-cystine I lalf -cystine Fragment moles per ‘105 9 weight in moles per mole of protein Daltons of protein ________~_~______--~~~~~________~-__------~~~~~~~~~------~~~~_~___~

Num her of s-s bridqcs

Fibrinogen

13.0

340 000 S,D

22

E

38.0

47 900 S,D

18.2

9

ECN BrI

55.2

33 000 eq

18.7

9

F CN 13r’

32.0

GO 000 cq

19.2

D

14.0

80300

D CN BrI

23.4

34 700 eq

8.0

FCN Br II

22.5

40 000 eq

9.0

44.0

S,D

9-10

11.2

6 4. 4-5

____________________~~~~~~~~~~~~~~------~~~~~~~~~~-----~~~~~~~~~~~~ (eq)

calculated

from

equilibrium

( S, D)

calculated

from

sedimentation

during

On

storage Fig.

of FCNBrI

even in

3 electrophoretic

I and E CNBrI derived CN Br are included for comparison.

half-cystine

does content

allows

at pH =

of previously

CNBr

cleavage

/16/.

3.5. /6/ studied

of fragments

been applied.

The

5 10 g

of

therefore

as cysteic

following

values

fibrinogen,

1) . It should

free

SH groups

fragments D and E

and DCN RrI have

/13-15/.

to estimate

In this and our previous

in each fragment. of cystine

(Table

coefficients

As can be seen F Cl\lBrII

not contain

determination

tine per

urea

to Y’phantis/8/

mobilities.

Fibrinogen

bridges

7M

from

according

and diffusion

patterns

D

identical

sedimentation

be added

F

acid 13.0,

according 32.0

Analysis

the number studies

of disulphide

chromatographic

to Schram

and 22.5

of the

et al.

moles

II respectively CNBrl and “CNBr that besides FCNBrI and FCNBrII

/9/ has

of half-cyswere

found

one more

DTSUI,I’lIIl~I~

Vo1.2,No.7

fragment lated.

containing

only about

Its molecular

size

Data concerning

‘I‘llCSL>

tl;llil

(15

CNRr Wc>ll

{IS

content

ItIc‘

VillUCS

01’

in bovine

IllC

IO5 g protein

was iso-

yet. fibrinogen,

main dcrivntivcs

ments are ri>mbined from the results /I,

per

has not been established

cleaved

,_ ‘77

I;‘RhGMI~:NTS

10 half-cystines

half-cystine

and E and in their I .

IITCII

are

of our present

presented size

~ll~~lc~C~ll~l1~

fragments

D

on Tnl~lc

ol’ LIIOSC l‘r;l~-

and previous

studies

3, 6/.

DIscussIoN __-_______________ results

‘l‘lle

ducts

presented

resulting

here

indicate

from CN I3r cleavage

ments of considerable

size

that out of the whole

of bovine

and rich

in S-S

FCN I~r I of about 60 000 Daltons

fragments FCNBrII

of about 40 000 Daltons

Our previous ting from

studies

plasmin

derivatives

/l,

proteolysis

( DCNnrI

6/ indicated

of bovine

of proof frag-

be isolated.

Namely

bridges

and

bridges. D and E resul-

that fragments

fibrinogen

contain

and ECNBrI)

could

and 9 disulphide

and 4 disulphide

3,

two types

fil)rinogcn

bonds

mixture

as well

considerable

CNRr

as their number

od S-S

bonds. Comparison lish

some

provide

relationship indications

As already

discussed

of bovine

after

N-terminal fected

sulphide sent also

bridges

treatment. bovine

in bovine

N-DSK

fragment

fragments

and may

molecule.

that probably

chains

only

the

in its electrophoretic

are

containing S-S

mobiwith

representing

of fibrinogen

9. Nine

E and in its derivative

in analogy

chains

to human N-DSK

contains

to estab-

represents

of its peptide

I3 /3andr

Zn contrast

FCNHrI

change

indicates

rich

1 allows

in fibrinogen

fragment

fragments

of the Acc,

on Table

disulphide

A drastic

the N-terminal

by CNnr

shown

localization

in the text

with thrombin

fragments

data

all these

to their

fibrinogen.

treatment

human N-DSK

and present between

some

N-DSK lity

of those

bridges

E CNBrI

/6/.

not af11 diare

pre-

It does

278

not seem N-DSK

chains

E CNBrI

that CNBr in bovine

it should

CNBrI’

E overlaps

with

C-terminal

from

only

position

1. Hence

than 4 methionines

If this is correct

beyond their

The fragment of fibrinogen. phide bridges. the CNBr

cleaved

4 S-S FCNBr

ced decrease

bridges

of disulphide

mass of peptides pcptidcs

our previous

E /l/ indicate

of polypeptide

in size

bridges

arc

E must be

off by CNBr

probably

chains

/lo, 17,18/,

simple access

is open for

Work

in fibrinogen

in this direction

close

another

disulphide

to produce

of fragment

D than E /l/

con-

/6/ . As shown on Fig .3 electrophoretic

in much further

E and a loss of 2 S-S

with considerably

higher

. As can be seen from

found in two fragments

for one of

by us DCNBrI

identical results

rich part

and 4-5 disul-

to the data obtained

D, designated

D with CNBr

well

has

/19/.

of 34 700 Daltons

appear

less

of fibrinogen

size of about 40 000 Daltons

of fragment

com-

a dimer compo-

of one or two chains

are very

tlcrivctl

in average

is apparently

to represent

than in case

in fragment

cleaved

that it contains

from N-terminal.

of fragment

corresponds

of fragment

data on the amino acid

and relatively

per molecule

II and D CNBrI

1) . This

methionines

values

namely

amounting to 15 000 Daltons

E. These

II appears

products

Treatment

patterns.

(33 000)

since

It has a molecular These

fragments

size bctwccn

at this meeting

FCNBr

but actually

in molcculnr

a convenient

been presented

in

The diffcrcncc

a total

methionine

part

bridges

only C-terminal

amino acid sequence

first

of S-S

can cleave

per mole and its molecule

of fragments

study on further

in each of these

that cleava-

.of E with CNBr

and ECNBrI

fragment

into account

the number

the N-terminal

chains.

one or two chains

sed of 3 pairs

present

part of frngmcnt

of bovine

already

are

therefore,

of any of the pep-

that not only the same number

and treatment

represent,

parts

and taking

does not dccrcnse

bonds

E (48 000)

from

(Table

v01.2,~0.3

fragment

N-terminal

( FCNBrl)

N-DSK

of its peptidc

fragment

not affect

be concluded

in N-DSK

fragments

both

does

E and ECNBr

located

taining

since

E by CNRr

the same disulphide

would

coincidence

FRAGMENTS

/4/.

ge of fragment

F

RICH

to be a more

Assuming tide

DISULPHIDE

bridges

number Table

D and one E which

advan-

of

1 the sum

derive

from

one

molecule

cf

fibrinogen

amounts

to 21 as compared

tc

total

22 found

in fil)rinogcn. All these

data lead us to an assumption

ment D. This

would

0 CN L3r1*) rich

mean that besides

in disulphide

that FCN DrII overlaps

N-DSK

bridges

two symmetrical

may be formed

with frag-

fragments

I)y CNI3r

cleavage

of

fibrinogen. Independent

studies

derived

from

In spite

of species

human fibrinogen

size

cleaved tcnce

f13-DSK.

3

other

in the formation apper

Daltons. mentioned

or three

fibrinogen,

weight

apparently

also

observed

10 half-cystines

its molecular

The term

disulphide

size

more

appropriate

polypcptide

chains

One might doubt,

ges.

fragments 201.

containing

Especially,

ments D are F CNHrII

located

from

to be at least

bovine in great

10

one for

a fragment

bound togcthcr

however,

sole

paper

fibrinogen

34 000

As already another from

fragbovine

yet.

proposed

cleaved

by Rlomback

off by CNBr.

of molecule

it should

bond as proposed

containing

be applied

/21/ some evidence regions

and Ill-DSK

part if not comp1etel.y

It seems two cr brid-

at al.

/lo,

one /lo/.

is provided

of fibrinogen

that fragmolecule.

from human fibrinogen located

et

to small

by Gardlund

bond is an intrachain

in the C-terminal

and II l-DSK

was

by some number of disulphidc

whether

1 S-S

which

product

is produced

has to be established

and

D results

largest

to F Ch’BrII

g protein

of fibrinogen

when this

In the accompanying

per

5

Il2-DSK

immunologically.

that in addition

the exis-

products”

The

of 33 000 Daltons

part

only

III-DSK,

containing

identical

results. pat-

recognize

of human fragment

knot has been originally

/2/ for the N-terminal

to be a very

authors

to H 1, H2 and H3-DSK.

The two were we have

disulphide

similar

, Iilra~C l‘r;l~qmclll s

mail1

nnmcly,

cleavage

very

knots”

in electrophoretic

ii1 IIlc

these

fibrinogen

in

D provide

similarities

N-DSK

to them CNnr

to have molecular

ment containing

al.

of DSK

similar

/ 10, 201 on “disulphide

c0illCl~l

Besides

of “two

to be very

estimated

types

According

striking

ai~tl tlisllIl)llitlc

by CN Br occur. of

et al.

and fragment

difference

111l~Iccul;lr.

ICI‘IIS,

of Gardlund

in the respective

Since appear frag-

DISULPHIDE

280

ments a more

D we propose descriptive

As mentioned with chemical realize

to call

the term

cleavage

proteolysis

bridges

present

so condidered

by plasmin

in fibrinogen as “disulphide

fragments

resulting

(C-DSK)

by

.

in conjunction

It may be of

practically

These

of fibrinogen

strictly

high molecular

contain

knots”

knots

with CNBr.

final

/3/.

regions

been proposed

of fibrinogen D and E,

rich

disulphide

DSK has

Vo1.2,No.3

FRAGMENTS

disulphide

name C-terminal

that fragmnets

brinogen

these

RICH

weight

interest

products

to of fi-

all of the disulphide could

be therefore

from biological

al-

cleavage

of

fibrinogen.

REFERENCES ___________________

1.

DUDEK,

G.A.

,K,LOCZEWlAK,

Z.S . and KOPEC, cular end products chem.Biophys.Acta:

M.,

BUDZYNSKI,

A.Z.,

LATALLO,

M. Characterization and comparison of macromoleof fibrinogen and fibrin proteolysis by plasmin.Bio214, 44, 1970

2.

BLOMBACK, IWANAGA,

B., BLOMBACK, M., HENSCHEN,A., HESSEL, S , and WOODS, K. R. N-terminal disulphide knot of Nature: 218, 130, 1968 human fibrinogen.

3.

LATALLO, Z.S, DUDEK, lation between the location disulphide 13,

37,

knots

B.,

G.A. and KLOCZEWIAK, M. A possible reof D and E fragments and of the N-terminal

in fibrinogen

molecule.

S cand.

J . Haematol.

: Suppl.

1971

4.

MARDER , V . J. Identification and purification of fibrinogen degradaconsiderations on the structure of tion products produced by plasmin; fibrinogen. Stand. J.Haematol. : Suppl. 13, 21, 1971

5.

SHULMAN, N. R. and CARROL, W. R. High molecuMARDER, V.J., lar weight derivatives of human fibrinogen produced by plasmin. Physiochemical and immunological characterization. J. Biol. Chcm. : 3, 2111,

6.

1969

DUDEK-WOJClECllOWSKA, G.A., KLOCZEWlAK, M., LATALL0,Z.S. and KOPEC, M. Characterization of large fragments rich in disulphide bridges from CNBr treated products of exhaustive proteolysis of fiBiochem. Biophys . Acta: 295, 536, 1973 brinogen by plasmin.

DISULPHIDE

Vol.Z,No.3

7. C’AIINMAN, risation

ll.J.,

ARNC~N,

of active

with cyanogen

8. YPHRNTIS

fragments

bromide.

, D.A.

Biochemistry:

9. SCHRAM,

It.

281

RICH FRAGMENTS

M . Iso~;I~ ion iintl

i111(1 SI
CII~~:ICI

c’-

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RICH FRAGMENTS

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KOWALSKA-LOTH, B., GRONDAHL, N. and disulphide knots in human fibrinogen.

, B. Hydrophobic

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KOPEC, M., Kl_OC%EWIAK, on the “double

lntcrnational

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Ds 11ington

TEISSEYRE, E., DUDEK-WOJCIECHOWSKA, PANKIEWlCZ, A. and LATALLO, Z.S. M., D” fragment from stabilized bovine fibrin (this

G., Studies issue)