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Plasmic degradation of fibrinogen: The preparation of a low molecular weight derivative of Fragment D
THROMBOSIS RESEARCH Printed in the United States
~01.3,
PP.
553-564,
1973
Pergamon Press, Inc.
PLASMIC DEGRADATION OF FIBRINOGEN: THE PREPARATION OF A LOW MOLECULAR WEIGHT DERIVATIVE OF FRAGMENT D Graham Kemp, Miha Furlan and Eugene A. Beck. Central Hematology Laboratory, Inselspital, and University of Berne, School of Medicine, Berne, Switzerland. (Received11.7.1973;In revisedform 24.9.1973. Acceptedby Editor B. Blomblck.)
ABSTRACT The conditions for the formation of a low molecular weight Fragment D (Fragment d) have been elucidated. Fragment D undergoes an irreversible conformational change on incubation at pH values below 3.5. This change leaves Fragment D more susceptible to additional plasmic digestion. Methods for the purification of Fragments E, D and d are described.
INTRODUCTION The structure of fibrinogen degradation products produced by the action of plasmin has been extensively studied. The reported molecular size of Fragment D (l), however, is variable, having been ascribed values ranging from 45,000 to over 100,000 (2 - 8). Some of this size heterogeneity could be due to varying degrees of plasmin digestion, but it has also been suggested that Fragment D is a molecular fragment of rather homogeneous size with eight charge isomers (9). Heterogeneity in the fibrinogen molecule itself has also been reported (lOj, and the action of a non-plasmin, thrombin-like component 553
FRAGMENTS D, d AND E
554
Vo1.3,No.5
suggested (11). In a recent publication from this laboratory (12), the isolation from Fragment D by CM-cellulose chromatography of a 45,000 molecular weight fraction was described. This was called Fragment d and is probably identical with the 45,000 Fragment D isolated by Mills and Triantaphyllopoulos (3). Mills (6) noted that after extensive plasmin digestion of fibrinogen in the presence of 2 M urea, the result was a Fragment D of molecular weight 43,000. This paper describes experiments which define the conditions under which Fragment d is produced, and describes methods developed for the separation of Fragments D and d, free from E. MATERIALS AND METHODS Human fibrinogen (KABI Grade L) was degraded by two different methods. 1. Endogenous plasmic digestion.
The endogenous plasmin
present in the fibrinogen was activated by streptokinase (Streptase, Behring, 100 units/ml final concentration). The reaction was stopped after 3 h or 24 h at 37OC, by the addition of Trasylol (Bayer) to a concentration of 100 units/ml and the mixture lyophilised. 2. Digest with purified plasmin.
Plasminogen-free fibrino-
gen, prepared by DEAE-cellulose chromatography (13) was digested at 370C for 24 h by activating plasminogen*(added in an enzyme-substrate ratio of 1 : 20., w/w) with streptokinase. The reaction was stopped by the addition of Trasylol, and the digestion mixture lyophilised.
* Purified human plasminogen, prepared by affinity chromatography, was kindly supplied by Dr. E.E. Rickli, Theodor Kocher Institute, Bern.
FRAGMENTS
Sephadex carried
G-200 Superfine
out as previously
system suggested
0.2 M &-amino
formate.
and
(Whatman CM-52) chromato-
out using buffer
systems based on 0.05 M
Columns were equilibrated
pH 3.0 or 4.25, and samples dialysed the equilibration
0.025 M Tris/HC :1,
(2) - namely
acid.
(CM) cellulose
graphy was carried ammonium
et al.
was
(5) using the buffer
1.0 M NaCl, 0.028 M sodium citrate
caproic
Carboxymethyl
555
(Pharmacia) gel filtration
described
by Marder
pH 7.4, containing
D, d AND E
pH before
at either
for at least 3 h at
application.
After
sample appli-
cation,
the column was eluted with 50 ml of equilibration
buffer,
and then with the same buffer
adjusted
to pH 7.5
with NH40H. In experiments
to determine
samples containing
bility,
(0.5 mg/ml
the effect of pH on solu-
a mixture
total concentration,
24 h endogenous
plasmic
digest
at pH 7.4, were dialysed
separated
values
incubation
between
Fragment
D isolated
matography
chloramphenicol.
described
out on digest
at pH 7.4, and CM-cellulose
The varying
chro-
contained
(pH 7.5) and
conditions
used are
in Table 1.
The variation followed
at 280 nm.
plasmin
D in 0.05 M sod'ium citrate
to pH
which remained
with plasmin was carried
(starting pH 4.25). All experiments
5 mg/ml Fragment 20 pg/ml
the samples were adjusted
from a 3 h endogenous
by G-200 chromatography
pH
with HCl or NH4OH as appro-
from the optical density
digestion
G-200
incubation
4.7 and 8.0, and the percentage
soluble was determined Additional
on Sephadex
from a
Buffers were made up from 0.05 M
formic acid, and the pH adjusted After
D and E
in a 2:l molar ratio)
at the appropriate
for 6 h at room temperature.
priate.
of Fragments
in the extinction
at room temperature
photometer.
at 280 nm with time was
using a Beckman
At zero time a 0.5 mg/ml
solution
DB-GT
spectro-
of Fragment
D
FRAGMENTS D, d AND E
556
vo1.3,No.5
TABLE 1 Additional Digestion with Plasmin: Sample Composition
2 M Urea
Plasminl
Trasylo12
Acid Pretreatment3
a. b.
+
C.
+
+
d. e.
+
+
+ +
f.
+ +
g*
Plasminogen was added in an enzyme/substrate ratio of 1 : 20 and activated with streptokinase. Added at the beginning of the incubation period (500 units/ml). Dialysis at pH 2.8 (ammonium formate) for 3 h.
in distilled water was adjusted to pH 2.8 with formic acid. A tyrosine solution of identical initial extinction at 280 nm was used as the reference. Immunoelectrophoresis, immunodiffusion and SDS-polyacrylamide gel electrophoresis were carried out as previously described (5, 12). RESULTS AND DISCUSSION Figures 1 a and b show the effect of preincubating a mixture of Fragments D and E in the pH range of 1.5 to 4.5, on the solubility between pH 4.7 and 8.0. Polyacrylamide gel electrophoresis showed that Fragment E remained soluble
FRAGMENTS D, d AND E
Vo1.3,No.5
557
throughout, while Fragment D could be completely precipitated after preincubation at pH 3.0 and below. The precipitated Fragment D was readily soluble in urea concentrations above 2 M and in 0.1 % SDS. Minimum solubility of Fragment D, after acid treatment, occurred between pH 5 and pH 7 (Fig. 1 a). A plot of solubility at pH 5.3 against the preincubation pH (Fig. 1 b), showed a sharp transition around pH 3.5 indicating a profound structural modification.
80 _
I260, 51 sLO_ FIG. 1 (a) Solubility of a Fragment D and E mixture after incubation 4 for 6 h at pH 4.0 (o-o) 80 pH 3.5 (+-+) and pH 3.0 (A-A) lb1 (b) Solubility of a Fragment D and E mixture at pH 5.3 after incubation for 6 h at varying pH values.
20 -
0 4,
I
I
5.0
6.0
7.0
PH .
??
.? ?
01 II
o-o-
2.0
80
LO
50
INCUBATIONpti
The extinction of a solution of Fragment D changed rapidly on adjustment of the pH to 2.8 (Fig. 2). The change in the structure of Fragment D, which caused the precipitation on raising the pH, is also rapid, being complete within 10 minutes.
FRAGMENTS D, d AND E
558,
Vo1.3,No.5
-IlO! i,
-0Dd ;.
3,
2,
-0.0
I.
I
10
20
30
4
40
llME( mid FIG. 2 The change, with time, in the extinction at 280 nm, of a 0.5 mg/ml solution of Fragment D, adjusted to pH 2.8 at zero time.
The results of SDS-polyacrylamide electrophoresis of the non-reduced products of extended plasmic digestion of Fragment D are summarised in Table 2. These results suggest that, without some unfolding of the D molecule, the digestion of Fragment D does not proceed to any significant extent beyond a fragment of mobility corresponding to a molecular weight of 73,000 (Table 2). In the presence of 2 M urea the digestion does not seem to proceed beyond an end point where the major product has a mobility corresponding to a molecular weight of 45,000. In addition, there are at least two species below 15,000. The digestion of acid-incubated Fragment D, however, is not identical. It also gives species with the mobilities expected for molecular weights of 38,000 and 35,000.
FRAGMENTS
vo1.3,No.5
TABLE
D, d AND E
559
2
Apparent Molecular Weights of the Products Resulting from the Additional Plasmic Digestion of Fragment D.
Molecular a. 1
94 >83 >>73
b.
94 >83 = 73 = 65
C.
94 >>83 >73
weight'
x 10'3
>14 = 12
d.
73 >>65 = 45
= 14 = 12
e.
45
= 14 = 12
f.
45 >38 >35 = 14 = 12
4.
94 >83 >>73
1
Details
are given in the text, and in Table
1.
2 In the interest of clarity, the same gel concentration (10 %) was used throughout. As pointed out by Neville (14) a linear relationship between mobility and the logarithm of molecular weight exists only between values of 15,000 and 70,000 in this system. Values quoted outwith this range are given as an indication of relative, and not absolute, size. Samples were not reduced.
There without
is ,also strong evidence the addition
of plasmin,
of additional particularly
sence of 2 M urea. This degradation the incorporation at zero time presence
of Trasylol
had been purified matography.
into the incubation
enzyme
that the 45,000 fragment
D which
is residual
and immunodiffusion produced
the
and CM-cellulose
The most likely candidate
chro-
plasmin.
studies
by additional
by
mixture
suggest
in a Fragment
by gel filtration
Immunoelectrophoresis
in the pre-
can be prevented
(Table 2 b and c). This would
of a contaminating
digestion
showed
digestion
FRAGMENTS D, d AND E
560
Vo1.3,No.5
with plasmin, either in 2 M urea or after acid incubation, is immunologically identical with the Fragment d produced by CM-cellulose chromatography (12). It gives a reaction of partial identity with Fragment D. In addition, immunoelectrophoresis and immunodiffusion studies, after incubation in the presence of 2 M urea, but without plasmin (Table 1 b), showed that the products contained material immunologically indistinguishable from Fragment d. As shown in Table 2, digestion had not proceeded beyond a fragment with an apparent molecular weight of 65,000 in this experiment.
-2s1.
PH
1 . 1.5
!! 8
I. 1.0
0.5 .
0
200
400 VOLUME
800
800
(ml1
FIG. 3 CM-cellulose chromatography of a D and E mixture on a 20 x 1.5 cm column. Starting pH, 4.25. O.D. at 280 nm, -_ pH, . . ... . .
above, showed that the incubation of Fragment D at pH values below 3.5 for a short time The observationqdescribed
vo1.3,No.5
FRAGMENTS D, d AND E
561
caused a change in its solubility properties at near neutral PH, and that even a purified Fragment D contained a hydrolytic
of a 45,000 molecular weight compo-
nent on SDS-polyacrylamide gel electrophoresis, nor any immunological evidence of Fragment d. Rechromatography of this fraction on a CM-cellulose column equilibrated at pH 3.0 (Fig. 4), resulted in the elution of Fragment d, 1.t
PH ,8.0 _70 -60 _ 50
. 4.0
n Y
250
500
!A!
3.0
750
VOLUME hl)
FIG.
4
Rechromatography of the second peak (Fragment D) from the separation shown in Fig. Son a CM-cellulose coliunn, 12 x 1.5 cm. Starting pH 3.0. 0-D. 280 nm. .._.._.__._.__ pH_
562
FRAGMENTS D, d AND E
Vo1.3,No.5
although low in yield (approximately 17 %, w/w). The remainder of the applied material could only be eluted with 6 M urea and 0.5 M NaOH. The elution pattern of Fragment d exhibits considerable tailing, and elution does not occur until the effluent pH is near neutral. The reason for the isolation of Fragment d by CM-cellulose chromatography is not clear. It is possible that the formation of this smaller fragment results from the action of a contaminating proteolytic agent on Fragment D, rendered susceptible by the pretreatment at pH 3.0. Similar results were obtained with a starting material prepared by endogenous plasmic digestion, and by digestion with purified plasmin. The report by Mills and Triantaphyllopoulos (3), that the Fragment D isolated by their procedure had a molecular weight of 45,000, is probably explained by the fact that the initial digestion was stopped by the addition of urea, and the mixture then dialysed at neutral pH and treated extensively at pH 3.0 prior to the column separation. In a previous publication (12), the suggestion was made that the formation of Fragment d was not a result of plasmic proteolysis. In the light of the findings reported here, this conclusion must be modified. The end point in the digestion of Fragment D by plasmin appears to be a species with an apparent molecular weight of 73,000. In the presence of urea, or after treatment at pH values below 3.5, digestion by plasmin will proceed further, to a stage where the major end product has a molecular weight of 45,000. We conclude that Fragment d results from the action of plasmin on conformationally modified Fragment D.
ACKNOWLEDGEMENTS The authors are indebted to Dr. E. Mihalyi for many help-
FRAGMENTS
Vo1.3,No.5
ful discussions.
The skillful
563
D, d AND E
technical
assistance
of
Miss A. Schmid and Miss L. Siebenhtiner is gratefully
acknow-
ledged. This work was supported No. 3.703.73
in part by research
from the Swiss National
grant
Foundation.
REFERENCES 1. Nussenzweig, V., Seligmann, M., Pelmont, J. and Grabar, P. Les produits de degradation du fibrinogene humain par la physico-chimiques. plasmine. I. Separation et propri&es Ann.Inst.Pasteur: 100, 377, 1961. High mole2. Marder, V.J., Shulman, N.R. and Carroll, W.R. cular weight derivatives of human fibrinogen produced by plasmin. I. Physicochemical and immunological characterisation. J.Biol.Chem.: 244, 2111, 1969. Distribution 3. Mills, D.A. and Triantaphyllopoulos, D.C. of carbohydrate among the polypeptide chains and plasmin digest products of human fibrinogen. Arch.Biochem.Biophys.: 135, 28, 1969. A structural aspect of human 4. Gaffney, P.J. and Dobos, P. fibrinogen suggested by its plasmin degradation. FEBS. Lett.: 15, 13, 1971. 5. Furlan, M. and Beck, E.A. Plasmic degradation of human fibrinogen. I. Structural characterisation of degradation products. Biochim.Biophys.Acta: 263, 631, 1972. 6. Mills, D.A. A molecular model for the proteolysis of human fibrinogen by plasmin. Biochim.Biophys.Acta: 263, 619, 1972. 7. Pizza, S.V., Schwartz, M.L., Hill, R.L. and McKee, P.A. The effect of plasmin on the subunit structure of human fibrinogen. J.Biol.Chem.: 247, 636, 1972. 8. Gsrdlund, B., Kowalska-Loth, B., Grijndahl, N.J. and Blomback, B. Plasmic degradation products of human fibrinogen. I. Isolation and characterisation of Fragments E and D and their relation to "disulfide knots". Thrombosis Research: l_, 371, 1972. 9. Catanzaro, A., Hathaway, G., Strathern, J. and Edgington, T. Structure and in vivo behaviour of fibrinogen Fragment D. Proc.Soc.Exp.Biol.Med.: 139, 1401, 1972.
564
FRAGMENTS D, d ANDE
vo1.3,No.5
Heterogeneity of human fibrinogen. 10. Gaffney, P.J. Nature: 230, 54, 1971. The non-plasmin proteolytic 11. Mills, D. and Karpatkin, S. origin of human fibrinogen heterogeneity. Biochim.Biophvs. Acta: 251, 121, 1971. Plasmic degradation of human 12. Furlan, M. and Beck, E.A. fibrinogen. II. Further characterisation of Fragment D. Biochim.Biophys.Acta: 310, 205, 1973. Chromatographic purification of fibrino13. Finlayson, J.S. gen. In: Fibrinogen, K. Laki (Ed), New York. Marcel Dekker Inc., 1968, p. 39. Molecular weight determination of protein14. Neville, D.M. dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J.Biol.Chem.: 246, 6328, 1971.
~01.3,
PP.
553-564,
1973
Pergamon Press, Inc.
PLASMIC DEGRADATION OF FIBRINOGEN: THE PREPARATION OF A LOW MOLECULAR WEIGHT DERIVATIVE OF FRAGMENT D Graham Kemp, Miha Furlan and Eugene A. Beck. Central Hematology Laboratory, Inselspital, and University of Berne, School of Medicine, Berne, Switzerland. (Received11.7.1973;In revisedform 24.9.1973. Acceptedby Editor B. Blomblck.)
ABSTRACT The conditions for the formation of a low molecular weight Fragment D (Fragment d) have been elucidated. Fragment D undergoes an irreversible conformational change on incubation at pH values below 3.5. This change leaves Fragment D more susceptible to additional plasmic digestion. Methods for the purification of Fragments E, D and d are described.
INTRODUCTION The structure of fibrinogen degradation products produced by the action of plasmin has been extensively studied. The reported molecular size of Fragment D (l), however, is variable, having been ascribed values ranging from 45,000 to over 100,000 (2 - 8). Some of this size heterogeneity could be due to varying degrees of plasmin digestion, but it has also been suggested that Fragment D is a molecular fragment of rather homogeneous size with eight charge isomers (9). Heterogeneity in the fibrinogen molecule itself has also been reported (lOj, and the action of a non-plasmin, thrombin-like component 553
FRAGMENTS D, d AND E
554
Vo1.3,No.5
suggested (11). In a recent publication from this laboratory (12), the isolation from Fragment D by CM-cellulose chromatography of a 45,000 molecular weight fraction was described. This was called Fragment d and is probably identical with the 45,000 Fragment D isolated by Mills and Triantaphyllopoulos (3). Mills (6) noted that after extensive plasmin digestion of fibrinogen in the presence of 2 M urea, the result was a Fragment D of molecular weight 43,000. This paper describes experiments which define the conditions under which Fragment d is produced, and describes methods developed for the separation of Fragments D and d, free from E. MATERIALS AND METHODS Human fibrinogen (KABI Grade L) was degraded by two different methods. 1. Endogenous plasmic digestion.
The endogenous plasmin
present in the fibrinogen was activated by streptokinase (Streptase, Behring, 100 units/ml final concentration). The reaction was stopped after 3 h or 24 h at 37OC, by the addition of Trasylol (Bayer) to a concentration of 100 units/ml and the mixture lyophilised. 2. Digest with purified plasmin.
Plasminogen-free fibrino-
gen, prepared by DEAE-cellulose chromatography (13) was digested at 370C for 24 h by activating plasminogen*(added in an enzyme-substrate ratio of 1 : 20., w/w) with streptokinase. The reaction was stopped by the addition of Trasylol, and the digestion mixture lyophilised.
* Purified human plasminogen, prepared by affinity chromatography, was kindly supplied by Dr. E.E. Rickli, Theodor Kocher Institute, Bern.
FRAGMENTS
Sephadex carried
G-200 Superfine
out as previously
system suggested
0.2 M &-amino
formate.
and
(Whatman CM-52) chromato-
out using buffer
systems based on 0.05 M
Columns were equilibrated
pH 3.0 or 4.25, and samples dialysed the equilibration
0.025 M Tris/HC :1,
(2) - namely
acid.
(CM) cellulose
graphy was carried ammonium
et al.
was
(5) using the buffer
1.0 M NaCl, 0.028 M sodium citrate
caproic
Carboxymethyl
555
(Pharmacia) gel filtration
described
by Marder
pH 7.4, containing
D, d AND E
pH before
at either
for at least 3 h at
application.
After
sample appli-
cation,
the column was eluted with 50 ml of equilibration
buffer,
and then with the same buffer
adjusted
to pH 7.5
with NH40H. In experiments
to determine
samples containing
bility,
(0.5 mg/ml
the effect of pH on solu-
a mixture
total concentration,
24 h endogenous
plasmic
digest
at pH 7.4, were dialysed
separated
values
incubation
between
Fragment
D isolated
matography
chloramphenicol.
described
out on digest
at pH 7.4, and CM-cellulose
The varying
chro-
contained
(pH 7.5) and
conditions
used are
in Table 1.
The variation followed
at 280 nm.
plasmin
D in 0.05 M sod'ium citrate
to pH
which remained
with plasmin was carried
(starting pH 4.25). All experiments
5 mg/ml Fragment 20 pg/ml
the samples were adjusted
from a 3 h endogenous
by G-200 chromatography
pH
with HCl or NH4OH as appro-
from the optical density
digestion
G-200
incubation
4.7 and 8.0, and the percentage
soluble was determined Additional
on Sephadex
from a
Buffers were made up from 0.05 M
formic acid, and the pH adjusted After
D and E
in a 2:l molar ratio)
at the appropriate
for 6 h at room temperature.
priate.
of Fragments
in the extinction
at room temperature
photometer.
at 280 nm with time was
using a Beckman
At zero time a 0.5 mg/ml
solution
DB-GT
spectro-
of Fragment
D
FRAGMENTS D, d AND E
556
vo1.3,No.5
TABLE 1 Additional Digestion with Plasmin: Sample Composition
2 M Urea
Plasminl
Trasylo12
Acid Pretreatment3
a. b.
+
C.
+
+
d. e.
+
+
+ +
f.
+ +
g*
Plasminogen was added in an enzyme/substrate ratio of 1 : 20 and activated with streptokinase. Added at the beginning of the incubation period (500 units/ml). Dialysis at pH 2.8 (ammonium formate) for 3 h.
in distilled water was adjusted to pH 2.8 with formic acid. A tyrosine solution of identical initial extinction at 280 nm was used as the reference. Immunoelectrophoresis, immunodiffusion and SDS-polyacrylamide gel electrophoresis were carried out as previously described (5, 12). RESULTS AND DISCUSSION Figures 1 a and b show the effect of preincubating a mixture of Fragments D and E in the pH range of 1.5 to 4.5, on the solubility between pH 4.7 and 8.0. Polyacrylamide gel electrophoresis showed that Fragment E remained soluble
FRAGMENTS D, d AND E
Vo1.3,No.5
557
throughout, while Fragment D could be completely precipitated after preincubation at pH 3.0 and below. The precipitated Fragment D was readily soluble in urea concentrations above 2 M and in 0.1 % SDS. Minimum solubility of Fragment D, after acid treatment, occurred between pH 5 and pH 7 (Fig. 1 a). A plot of solubility at pH 5.3 against the preincubation pH (Fig. 1 b), showed a sharp transition around pH 3.5 indicating a profound structural modification.
80 _
I260, 51 sLO_ FIG. 1 (a) Solubility of a Fragment D and E mixture after incubation 4 for 6 h at pH 4.0 (o-o) 80 pH 3.5 (+-+) and pH 3.0 (A-A) lb1 (b) Solubility of a Fragment D and E mixture at pH 5.3 after incubation for 6 h at varying pH values.
20 -
0 4,
I
I
5.0
6.0
7.0
PH .
??
.? ?
01 II
o-o-
2.0
80
LO
50
INCUBATIONpti
The extinction of a solution of Fragment D changed rapidly on adjustment of the pH to 2.8 (Fig. 2). The change in the structure of Fragment D, which caused the precipitation on raising the pH, is also rapid, being complete within 10 minutes.
FRAGMENTS D, d AND E
558,
Vo1.3,No.5
-IlO! i,
-0Dd ;.
3,
2,
-0.0
I.
I
10
20
30
4
40
llME( mid FIG. 2 The change, with time, in the extinction at 280 nm, of a 0.5 mg/ml solution of Fragment D, adjusted to pH 2.8 at zero time.
The results of SDS-polyacrylamide electrophoresis of the non-reduced products of extended plasmic digestion of Fragment D are summarised in Table 2. These results suggest that, without some unfolding of the D molecule, the digestion of Fragment D does not proceed to any significant extent beyond a fragment of mobility corresponding to a molecular weight of 73,000 (Table 2). In the presence of 2 M urea the digestion does not seem to proceed beyond an end point where the major product has a mobility corresponding to a molecular weight of 45,000. In addition, there are at least two species below 15,000. The digestion of acid-incubated Fragment D, however, is not identical. It also gives species with the mobilities expected for molecular weights of 38,000 and 35,000.
FRAGMENTS
vo1.3,No.5
TABLE
D, d AND E
559
2
Apparent Molecular Weights of the Products Resulting from the Additional Plasmic Digestion of Fragment D.
Molecular a. 1
94 >83 >>73
b.
94 >83 = 73 = 65
C.
94 >>83 >73
weight'
x 10'3
>14 = 12
d.
73 >>65 = 45
= 14 = 12
e.
45
= 14 = 12
f.
45 >38 >35 = 14 = 12
4.
94 >83 >>73
1
Details
are given in the text, and in Table
1.
2 In the interest of clarity, the same gel concentration (10 %) was used throughout. As pointed out by Neville (14) a linear relationship between mobility and the logarithm of molecular weight exists only between values of 15,000 and 70,000 in this system. Values quoted outwith this range are given as an indication of relative, and not absolute, size. Samples were not reduced.
There without
is ,also strong evidence the addition
of plasmin,
of additional particularly
sence of 2 M urea. This degradation the incorporation at zero time presence
of Trasylol
had been purified matography.
into the incubation
enzyme
that the 45,000 fragment
D which
is residual
and immunodiffusion produced
the
and CM-cellulose
The most likely candidate
chro-
plasmin.
studies
by additional
by
mixture
suggest
in a Fragment
by gel filtration
Immunoelectrophoresis
in the pre-
can be prevented
(Table 2 b and c). This would
of a contaminating
digestion
showed
digestion
FRAGMENTS D, d AND E
560
Vo1.3,No.5
with plasmin, either in 2 M urea or after acid incubation, is immunologically identical with the Fragment d produced by CM-cellulose chromatography (12). It gives a reaction of partial identity with Fragment D. In addition, immunoelectrophoresis and immunodiffusion studies, after incubation in the presence of 2 M urea, but without plasmin (Table 1 b), showed that the products contained material immunologically indistinguishable from Fragment d. As shown in Table 2, digestion had not proceeded beyond a fragment with an apparent molecular weight of 65,000 in this experiment.
-2s1.
PH
1 . 1.5
!! 8
I. 1.0
0.5 .
0
200
400 VOLUME
800
800
(ml1
FIG. 3 CM-cellulose chromatography of a D and E mixture on a 20 x 1.5 cm column. Starting pH, 4.25. O.D. at 280 nm, -_ pH, . . ... . .
above, showed that the incubation of Fragment D at pH values below 3.5 for a short time The observationqdescribed
vo1.3,No.5
FRAGMENTS D, d AND E
561
caused a change in its solubility properties at near neutral PH, and that even a purified Fragment D contained a hydrolytic
of a 45,000 molecular weight compo-
nent on SDS-polyacrylamide gel electrophoresis, nor any immunological evidence of Fragment d. Rechromatography of this fraction on a CM-cellulose column equilibrated at pH 3.0 (Fig. 4), resulted in the elution of Fragment d, 1.t
PH ,8.0 _70 -60 _ 50
. 4.0
n Y
250
500
!A!
3.0
750
VOLUME hl)
FIG.
4
Rechromatography of the second peak (Fragment D) from the separation shown in Fig. Son a CM-cellulose coliunn, 12 x 1.5 cm. Starting pH 3.0. 0-D. 280 nm. .._.._.__._.__ pH_
562
FRAGMENTS D, d AND E
Vo1.3,No.5
although low in yield (approximately 17 %, w/w). The remainder of the applied material could only be eluted with 6 M urea and 0.5 M NaOH. The elution pattern of Fragment d exhibits considerable tailing, and elution does not occur until the effluent pH is near neutral. The reason for the isolation of Fragment d by CM-cellulose chromatography is not clear. It is possible that the formation of this smaller fragment results from the action of a contaminating proteolytic agent on Fragment D, rendered susceptible by the pretreatment at pH 3.0. Similar results were obtained with a starting material prepared by endogenous plasmic digestion, and by digestion with purified plasmin. The report by Mills and Triantaphyllopoulos (3), that the Fragment D isolated by their procedure had a molecular weight of 45,000, is probably explained by the fact that the initial digestion was stopped by the addition of urea, and the mixture then dialysed at neutral pH and treated extensively at pH 3.0 prior to the column separation. In a previous publication (12), the suggestion was made that the formation of Fragment d was not a result of plasmic proteolysis. In the light of the findings reported here, this conclusion must be modified. The end point in the digestion of Fragment D by plasmin appears to be a species with an apparent molecular weight of 73,000. In the presence of urea, or after treatment at pH values below 3.5, digestion by plasmin will proceed further, to a stage where the major end product has a molecular weight of 45,000. We conclude that Fragment d results from the action of plasmin on conformationally modified Fragment D.
ACKNOWLEDGEMENTS The authors are indebted to Dr. E. Mihalyi for many help-
FRAGMENTS
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ful discussions.
The skillful
563
D, d AND E
technical
assistance
of
Miss A. Schmid and Miss L. Siebenhtiner is gratefully
acknow-
ledged. This work was supported No. 3.703.73
in part by research
from the Swiss National
grant
Foundation.
REFERENCES 1. Nussenzweig, V., Seligmann, M., Pelmont, J. and Grabar, P. Les produits de degradation du fibrinogene humain par la physico-chimiques. plasmine. I. Separation et propri&es Ann.Inst.Pasteur: 100, 377, 1961. High mole2. Marder, V.J., Shulman, N.R. and Carroll, W.R. cular weight derivatives of human fibrinogen produced by plasmin. I. Physicochemical and immunological characterisation. J.Biol.Chem.: 244, 2111, 1969. Distribution 3. Mills, D.A. and Triantaphyllopoulos, D.C. of carbohydrate among the polypeptide chains and plasmin digest products of human fibrinogen. Arch.Biochem.Biophys.: 135, 28, 1969. A structural aspect of human 4. Gaffney, P.J. and Dobos, P. fibrinogen suggested by its plasmin degradation. FEBS. Lett.: 15, 13, 1971. 5. Furlan, M. and Beck, E.A. Plasmic degradation of human fibrinogen. I. Structural characterisation of degradation products. Biochim.Biophys.Acta: 263, 631, 1972. 6. Mills, D.A. A molecular model for the proteolysis of human fibrinogen by plasmin. Biochim.Biophys.Acta: 263, 619, 1972. 7. Pizza, S.V., Schwartz, M.L., Hill, R.L. and McKee, P.A. The effect of plasmin on the subunit structure of human fibrinogen. J.Biol.Chem.: 247, 636, 1972. 8. Gsrdlund, B., Kowalska-Loth, B., Grijndahl, N.J. and Blomback, B. Plasmic degradation products of human fibrinogen. I. Isolation and characterisation of Fragments E and D and their relation to "disulfide knots". Thrombosis Research: l_, 371, 1972. 9. Catanzaro, A., Hathaway, G., Strathern, J. and Edgington, T. Structure and in vivo behaviour of fibrinogen Fragment D. Proc.Soc.Exp.Biol.Med.: 139, 1401, 1972.
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Heterogeneity of human fibrinogen. 10. Gaffney, P.J. Nature: 230, 54, 1971. The non-plasmin proteolytic 11. Mills, D. and Karpatkin, S. origin of human fibrinogen heterogeneity. Biochim.Biophvs. Acta: 251, 121, 1971. Plasmic degradation of human 12. Furlan, M. and Beck, E.A. fibrinogen. II. Further characterisation of Fragment D. Biochim.Biophys.Acta: 310, 205, 1973. Chromatographic purification of fibrino13. Finlayson, J.S. gen. In: Fibrinogen, K. Laki (Ed), New York. Marcel Dekker Inc., 1968, p. 39. Molecular weight determination of protein14. Neville, D.M. dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J.Biol.Chem.: 246, 6328, 1971.