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Amino acid sequence of the carboxy-terminal cyanogen bromide peptide of the human fibrinogen β-chain: Homology with the corresponding γ-chain peptide and presence in fragment D
617
Bioehimica e t Biophysica A cta, 386 (1975) 617--622 O Eisevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
B B A Report BBA 31188
AMINO ACID SEQUENCE OF THE CARBOXY-TERMINAL CYANOGEN BROMIDE PEPTIDE OF THE HUMAN FIBRINOGEN ~-CHAIN: HOMOLOGY WITH THE CORRESPONDING 7-CHAIN PEPTIDE AND PRESENCE IN FRAGMENT D
TA KAS HI T A K A G I and R U S S E L L F. D O O L I T T L E
Department of Chemistry, University of California, San Diego, La JoUa, Calif. 92037
(U.S.A.) (Received February 6th, 1975)
Summary The carboxy-terminal cyanogen bromide fragment of the human fibrinogen ~-chain has been isolated and its structure determined. It is a nonapeptide with the sequence Lys-Ile-Arg-Pro-Phe-Phe-Pro-Gln-Gln and is homologous with a portion of the carboxy-terminal cyanogen bromide fragment of the T-chain. The peptide has also been isolated in full yield from cyanogen bromide digests of the plasmin-derived fragment D, indicating that the carboxy-terminal region of the 13-chain is resistant to plasmin digestion. In contrast, a small portion of the corresponding T-chain carboxy-terminal region was missing in the same fragment D.
During the course of structural studies on the plasmin-derived fragment D of human fibrinogen, we isolated a small cyanogen bromide fragment which did not contain homoserine and thus represented one of the carboxyterminals of that moiety. We were surprise to find that the carboxy-terminal residue of this nonapeptide was glutamine, since the specificity of plasmin is primarily directed toward lysyl (and to a lesser extent toward arginyl) bonds [ 1]. Subsequent examination of the cyanogen bromide digestion products of the individual a-, ~- and 7-chains of human fibrinogen revealed, however, that intact ~-chains also have this peptide at their carboxy-terminus, in contrast to a previous report that the carboxy-terminal residue is valine [2]. Most interesting, however, was the fact that the peptide was homologous to a portion of the carboxy-terminal cyanogen bromide fragment of the fibrinogen T-chain-the sequence of which we have previously reported [3,4], suggesting a common evolutionary ancestry for the ~- and T-chains. Human fibrinogen was prepared according to previously described
618 procedures [5]. Fragment D was prepared by digestion with human plasmin (Kabi, 10 C.T.A. units/ml) in 50% glycerol. A 1% solution of fibrinogen in 0.15 M NaC1, 0.05 M Tris buffer, pH 7.5, was mixed with the plasmin solution in a ratio of 15:1 by volume. At the end of 15 h digestion at room temperature, the mixture was applied directly to a Sephadex G-100 column equilibrated with 10% acetic acid; full details of the isolation procedure are reported in another article [6]. The homogeneity and size of the fragment D thus isolated was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, both before and after reduction of disulfide bonds. The individual a-,/3- and 7-chains of human fibrinogen were prepared by sulfitolysis of the fibrinogen followed by chromatography on carboxy-methyl cellulose in the presence of 8 M urea [7]. The a- and ~-chain pools were subsequently re-chromatographed using the same system, in order to improve their separation. The chains were reduced and alkylated, subjected to cyanogen bromide digestion (70% formic acid, 16 h, room temperature), lyophilized, and then fractionated on a Sephadex G-50 column equilibrated with 10% acetic acid (Fig. 1). The cyanogen bromide digestion of fragment D was conducted silimarly, but fractionation of the fragments was begun instead on a Sephadex G-100 column (Fig. 2). In either case, small peptide material was purified further by paper electrophoresis at pH 2 and pH 5. Amino acid sequences were determined by classical procedures; detailed references are available in our earlier articles [6,8]. 8 0'6~ I '
V
c 0 0.4~ oo .~ 0.2
x 0 , 0 5b
70 90 110 FRACTION NUMBER
130
150
Fig. i . F r a c t i o n a t i o n of peptides obtained from c y a n o g e n bromide digestion of h u m a n fibrinogen ~-chain o n Sephadex G-50 (3 X 9 0 c m ) equilibrated and eluted with 1 0 ~ acetic acid. F l o w r a t e = 18 m l / h ; f r a c t i o n size = 3 m l .
The same nonapeptide lacking homoserine was isolated from cyanogen bromide digests of both human fibrinogen ~-chains and fragment D. In the case of the ~-chain digest, the carboxy-terminal peptide was found in peak IX (Fig. 1); the corresponding peptide from fragment D was found in peaks IV and V (Fig. 2). The yield of this carboxy-terminal peptide was about the same in both cases, matching the yields of the best recovered peptides in either situation (the uncorrected yields after a five-step isolation procedure exceeded 25%). The carboxy-terminal cyanogen bromide fragment of the T-chain, on the other hand, was isolated from the fragment D in an altered form (from peak III, Fig. 2), the pentapeptidyl sequence at the carboxyterminus having been clipped off during the plasmin digestion (Table I). A
619
summary of the amino acid sequence data and a comparison of the carho:vyterminal cyanogen bromide fragments of the ~- and 7-chains are presented in Fig. 3. ~1
t
i
1.0 E
I
C
o 0o cq
"8 o.£
411
0 20
40
60 FRACTION
80 100 NUMBER
120
Fig.2. F r a e t i o n a t i o n o f p e p t i d e s o b t a i n e d f r o m c y a n o g e n b r o m i d e d i g e s t i o n o f fratr,m e n t D o n S e p h a d e x G - I O 0 (3 X 7 0 c m ) e q u i l i b r a t e d a n d e l u t e d w i t h 10% a c e t i c acid. F l o w r a t e = 18 m l / h ; f r a c t i o n size = 3 ml.
The amino acid sequence of the ~-chain carboxy-terminal region is significant on a number of counts. F o r e m o s t among these is its h o m o l o g y with a segment of the carboxy-terminal region of the 7-chain, the implication being that the t w o chains are sprung from a c o m m o n ancestor. Previously the suggestion was made that the a- and/~-chains of vertebrate fibrinogen were descended from a c o m m o n ancestral form [9]. Subsequent amino acid sequence data obtained from an amino-terminal cyanogen bromide fragment ("disulfide k n o t " ) revealed that all three chains have homologous pentapeptidyl sequences in the neighborhood of t w o regularly spaced cysteine residues [ 1 0 ] , raising the possibility that the three polypeptides have descended from a c o m m o n ancestor. Although we have commented on the structure-function consequences of fibrinogen evolving from a set of equivalent chains [ 11 ], we were frankly prepared for the eventuality that easily perceived homologies among the fibrinogen chains might have been blurred b y excessive amino acid replacements during the course of vertebrate evolution. Now, however, it is our contention that the occurrence of five identities in a string of six residues near the carboxy-terminus strongly favors the notion o f j3- and 7-chains arising as the result of gene duplication (cf. ref. 12). We are n o t y e t in a position to judge whether or n o t this was a discrete duplication leading to separate chains, or a contiguous (tandem) duplication giving rise to a single-chained profibrinogen [ 11 ]. ' B e y o n d the significance of c o m m o n ancestry for the j3- and 7-chains, a number of other interesting features have been revealed b y this investigation. The fact that the carboxy-terminus of the ~-chain remains intact during plasmin digestion under the conditions described, whereas the carboxy-terminus of the 7-chain is subject to plasmin attack, is at variance with the digestive scheme as described in some previous reports [13,14] b u t supportive of others [ 1 5 ] . In this regard, the apparent molecular weight of the unreduced fragment D used in our studies was 85 000 as determined on sodium dodecyl
620
Ly~-~e--~rg--Pro--Phe--Phe--Pro--Gln--Glnl CH-2
t
•'
CH-1
CN-9
"l "I
~ • - • IMet--LYs--IlelArgl Pr°--Phe - Phe--Pro--Gln--GlnC ~f'" '1Met--Lys--Ile~Ile--IPro--Phe" -Asn--Arg--Leu--Thr--Ile--Gly--Glu--Gly -28
-26
-22
-24
-20
-18
-16
Plasmin
l
•
--Gln--Gln--His--His--Leu~G1F--Gly--Ala~Lys--Gln--Ala--Gly--Asp--V ale -14
-12
-10
-8
-6
-4
-2
Fig.3. A m i n o a c i d s e q u e n c e s o f t h e c a r b o x y - t e r m i n a l r e g i o n s o f h u m a n f i b r i n o g e n ~- a n d 7 - c h a i n s . T h e data used to establish the sequence of the carboxy-terminal cyanogen bromide fragment of the ~-chain are summarized in the upper left hand comer: CN = cyanogen bromide; Ch = chymotrypsin; = carboxypeptidue A; =. ffi d e t e r m i n a t i o n b y t h e D N S - P I T C p r o c e d u r e . 7 - c h a i n r e s i d u e s are numbered negatively from the carboxy-terminus. Homologous regions are boxed. The * marks t h e g l u t a m i n e c r o s s l i n k i n g a c c e p t e r site; a b o n d a t t a c k e d b y p l u m t n d u r i n g t h e f o r m a t i o n o f f r a g m e n t D is n o t e d n e x t t o t h e l y s i n e c r o s s l i n k i n g d o n o r site.
sulphate polyacrylamide gel electrophoresis; gels run under reducing conditions yielded apparent molecular weights for the a-, ~- and 7-chain portions of 10 000, 44 000 and 35 000 respectively. It is also of interest to note that the removal of the pentapeptidyl sequence from the carboxy-terminus of the 7-chain ought to render these D fragments incapable of 7 ~ dimer formation by factor XIII, since the lysine donor residue is now the carboxy-terminal amino acid. On the other hand, the fragment still ought to be able to incorporate substitute donors since the glutamine accepter residue has not been violated [3]. Finally, some comment ought to be made regarding a previous report that the carboxy-terminal residue of the human fibrinogen ~-chain is valine [ 2 ] The low yields reported in that study raise the possibility that the ~-chains used may have been contaminated with 7-chains, the carboxy-terminals of
2.3 (2) .
Alanine
27
1.0 (1)
22
0.8 (1)
1.4 (2) .
.
2.3 (2) .
.
. 1.4 (3) 1 . 9 (2)
0.7 (1) .
3 . 2 (3) 1.1 ( I ) 4.0 (4)
1 . 0 (1) 2 . 2 (2)
.
.
.
1 . 0 (1) 1 . 8 (2)
.
0.9 (I) 2.6 (3) 2.1 ( 2 )
(4) (I) (5)
1,3 (I) 1.0 (I)
.
.
CN-IIIb6 ( F r a g m e n t D)
.
.
.
9
0.8 (1)
1.0 (1)
1.8 (2) .
. 0 . 7 (1) .
.
. 2.2 (2) 2.4 (2) .
. .
.
.
.
.
CN-IX (~-Chain)
.
.
.
. .
9
0.9 (1)
1.0 (1) .
2 . 2 (2)
.
. 0.8 (1)
. .
. 2 . 2 (2) 1.9 (2)
.
.
.
. .
.
CN-IVb-2 ( F r a g m e n t D)
.
.
.
.
.
. .
I S O L A T E D F R O M H U M A N F I B R I N O G E N ~- A N D
4
--
--
0 , 7 (1)
--
2 . 3 (2) 1 . 0 (1)
~-CN-IX-CH-1
5
0.9
(1)
1.2 (1)
1 . 0 (1)
0 . 6 (1)
-1.3 ( 1 )
~-CN-IX-CH-2
Chymotryptic peptides
CYANOGEN BROMIDE FRAGMENTS
* V a l u e s are given in r e s i d u e s p e r m o l . S a m p l e s w e r e h y d r o l y z e d f o r 2 4 h in 5.7 M HCI a t I I 0 ° C a n d t h e n a n a l y z e d o n a S p i n e o A u t o m a t i c A m i n o A c i d A n a l y z e r e m p l o y i n g a t h r e e - b u f f e r single c o l u m n p r o c e d u r e , All p e p t i d e s w e r e t r y p t o p h a n - n e g a t i v e . * * I n t h e case o f f r a g m e n t 7 C N - I V t h e v a l u e s are t h e a v e r a g e o f 2 4 a n d 4 8 h h y d r o l y s e s . T h e n e - n e b o n d in t h e s e p e p t i d e 8 i8 pa~ticttla~|y t e s / s t a n t t o a c i d h y d r o l y s i s , a n d we h a v e p r e v i o u s l y s h o w n t h a t 7 2 h h y d r o l y s i s is r e q u i r e d f o r q u a n t i t a t i v e r e c o v e r y [ 4 ] . T h i s a c c o u n t s f o r t h e p o o r ~ecoverY o f Isole1~cine i n t h e 2 4 h h y d r o l y s i s o f C N - I I I b 6 o b t a i n e d f r o m f r a g m e n t D.
Total residues
Cysteine V-line Isoleueine** Leucine Tyrosine Phenylalanine Histidine Lysine Homoserine Arginine
2.2 I.I . 4.2 0.9 4.9
Aspartic acid Threonine Serine Glutamic acid Proline Glyeine
(2) (1)
CN-IV** (T-Chain)
Amino acid
AMINO ACID COMPOSITIONS OF CARBOXY-TERMINAL 7 - C H A I N S A N D F R O M F R A G M E N T D*
TABLE I
O~ b0
622
which are undisputably valine [3]. Beyond that, the difficulties of determining carboxy-terminal glutamine residues are great. Not only does hydrazinolysis not release a free amino acid, but the free glutamine residue released by carboxypeptidase A is prone to cyclize into the elusive pyrrolidone carboxylic acid. There is always the alternative possibility, of course, that our ~-chains are the product of some premature proteolysis, but the excellent yields achieved in all experiments tend to disallow such an explanation. In summary, amino acid sequence studies on some key peptides isolated from human fibrinogen fragment D have provided valuable information about the structure and evolution of fibrinogen as well as shedding light on the sequence of events which occurs during the plasmic degradation of that molecule. The excellent technical assistance of Barbara Cottrell, Jay Solnick and Marcia Riley is gratefully noted. These studies were supported by N.I.H. Grants HE-12,759 and GM-17,702. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Weinstein, M.J. and Doolittle, R.F. (1972) Biochim. Biophys. A e t a 258, 577--590. Okude, M. and Iwanaga, S. (1971) Biochim. Biophys. A c t a 251, 185--196. Chert, R. and Doolittle, R.F. (1971) Biochemistry 10, 4486--4491. Sharp, J.J., Cassman, K.G. and Doollttle, R.F. (1972) FEBS Lett. 25, 334--336. Doolittle, R.F., Schubert, D. and Schwartz, S.A. (1967) Arch. Biochem. Biophys. 118, 456--467. Takagi, T. and Doolittle, R.F. (1975) Biochemistry, in the press. Henschen, A. (1964) Ark. Kern. 22, 1--28. Taka~, T. and Doolittle, R.F. (1974) Biochemistry 13, 750--756. Doolittle, R.F. (1970) Thromb. Diath. Haem., Supp. 39, 25--42. Blomb~'ck, B. (1971) in Molecular Evolution. 2. Biochemical E v o l u t i o n and the Origin of Life (E. Schoffeniels, ed.), pp. 112--129, North Holland Publishing C o m p a n y , Amsterdam. Doolittle, R.F. (1973) Adv. Protein Chem., 27, 1--109. Singer, S.J. and Doolittle, R.F. (1966) Science 153, 13--25. Pizzo, S.V., Schwartz, M.L., Hill, R.L. and McKee, P.A. (1972) J. Biol. Chem. 247, 636--645. Mosesson, M.W., Finlayson, J.S. and Galanakis, D.K. (1973) J. Biol. Chem. 248, 7913--7929. Furlan, M. and Beck, E.A. (1972) Biochim. Biophys. A c t a 263, 631---644.
Bioehimica e t Biophysica A cta, 386 (1975) 617--622 O Eisevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
B B A Report BBA 31188
AMINO ACID SEQUENCE OF THE CARBOXY-TERMINAL CYANOGEN BROMIDE PEPTIDE OF THE HUMAN FIBRINOGEN ~-CHAIN: HOMOLOGY WITH THE CORRESPONDING 7-CHAIN PEPTIDE AND PRESENCE IN FRAGMENT D
TA KAS HI T A K A G I and R U S S E L L F. D O O L I T T L E
Department of Chemistry, University of California, San Diego, La JoUa, Calif. 92037
(U.S.A.) (Received February 6th, 1975)
Summary The carboxy-terminal cyanogen bromide fragment of the human fibrinogen ~-chain has been isolated and its structure determined. It is a nonapeptide with the sequence Lys-Ile-Arg-Pro-Phe-Phe-Pro-Gln-Gln and is homologous with a portion of the carboxy-terminal cyanogen bromide fragment of the T-chain. The peptide has also been isolated in full yield from cyanogen bromide digests of the plasmin-derived fragment D, indicating that the carboxy-terminal region of the 13-chain is resistant to plasmin digestion. In contrast, a small portion of the corresponding T-chain carboxy-terminal region was missing in the same fragment D.
During the course of structural studies on the plasmin-derived fragment D of human fibrinogen, we isolated a small cyanogen bromide fragment which did not contain homoserine and thus represented one of the carboxyterminals of that moiety. We were surprise to find that the carboxy-terminal residue of this nonapeptide was glutamine, since the specificity of plasmin is primarily directed toward lysyl (and to a lesser extent toward arginyl) bonds [ 1]. Subsequent examination of the cyanogen bromide digestion products of the individual a-, ~- and 7-chains of human fibrinogen revealed, however, that intact ~-chains also have this peptide at their carboxy-terminus, in contrast to a previous report that the carboxy-terminal residue is valine [2]. Most interesting, however, was the fact that the peptide was homologous to a portion of the carboxy-terminal cyanogen bromide fragment of the fibrinogen T-chain-the sequence of which we have previously reported [3,4], suggesting a common evolutionary ancestry for the ~- and T-chains. Human fibrinogen was prepared according to previously described
618 procedures [5]. Fragment D was prepared by digestion with human plasmin (Kabi, 10 C.T.A. units/ml) in 50% glycerol. A 1% solution of fibrinogen in 0.15 M NaC1, 0.05 M Tris buffer, pH 7.5, was mixed with the plasmin solution in a ratio of 15:1 by volume. At the end of 15 h digestion at room temperature, the mixture was applied directly to a Sephadex G-100 column equilibrated with 10% acetic acid; full details of the isolation procedure are reported in another article [6]. The homogeneity and size of the fragment D thus isolated was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, both before and after reduction of disulfide bonds. The individual a-,/3- and 7-chains of human fibrinogen were prepared by sulfitolysis of the fibrinogen followed by chromatography on carboxy-methyl cellulose in the presence of 8 M urea [7]. The a- and ~-chain pools were subsequently re-chromatographed using the same system, in order to improve their separation. The chains were reduced and alkylated, subjected to cyanogen bromide digestion (70% formic acid, 16 h, room temperature), lyophilized, and then fractionated on a Sephadex G-50 column equilibrated with 10% acetic acid (Fig. 1). The cyanogen bromide digestion of fragment D was conducted silimarly, but fractionation of the fragments was begun instead on a Sephadex G-100 column (Fig. 2). In either case, small peptide material was purified further by paper electrophoresis at pH 2 and pH 5. Amino acid sequences were determined by classical procedures; detailed references are available in our earlier articles [6,8]. 8 0'6~ I '
V
c 0 0.4~ oo .~ 0.2
x 0 , 0 5b
70 90 110 FRACTION NUMBER
130
150
Fig. i . F r a c t i o n a t i o n of peptides obtained from c y a n o g e n bromide digestion of h u m a n fibrinogen ~-chain o n Sephadex G-50 (3 X 9 0 c m ) equilibrated and eluted with 1 0 ~ acetic acid. F l o w r a t e = 18 m l / h ; f r a c t i o n size = 3 m l .
The same nonapeptide lacking homoserine was isolated from cyanogen bromide digests of both human fibrinogen ~-chains and fragment D. In the case of the ~-chain digest, the carboxy-terminal peptide was found in peak IX (Fig. 1); the corresponding peptide from fragment D was found in peaks IV and V (Fig. 2). The yield of this carboxy-terminal peptide was about the same in both cases, matching the yields of the best recovered peptides in either situation (the uncorrected yields after a five-step isolation procedure exceeded 25%). The carboxy-terminal cyanogen bromide fragment of the T-chain, on the other hand, was isolated from the fragment D in an altered form (from peak III, Fig. 2), the pentapeptidyl sequence at the carboxyterminus having been clipped off during the plasmin digestion (Table I). A
619
summary of the amino acid sequence data and a comparison of the carho:vyterminal cyanogen bromide fragments of the ~- and 7-chains are presented in Fig. 3. ~1
t
i
1.0 E
I
C
o 0o cq
"8 o.£
411
0 20
40
60 FRACTION
80 100 NUMBER
120
Fig.2. F r a e t i o n a t i o n o f p e p t i d e s o b t a i n e d f r o m c y a n o g e n b r o m i d e d i g e s t i o n o f fratr,m e n t D o n S e p h a d e x G - I O 0 (3 X 7 0 c m ) e q u i l i b r a t e d a n d e l u t e d w i t h 10% a c e t i c acid. F l o w r a t e = 18 m l / h ; f r a c t i o n size = 3 ml.
The amino acid sequence of the ~-chain carboxy-terminal region is significant on a number of counts. F o r e m o s t among these is its h o m o l o g y with a segment of the carboxy-terminal region of the 7-chain, the implication being that the t w o chains are sprung from a c o m m o n ancestor. Previously the suggestion was made that the a- and/~-chains of vertebrate fibrinogen were descended from a c o m m o n ancestral form [9]. Subsequent amino acid sequence data obtained from an amino-terminal cyanogen bromide fragment ("disulfide k n o t " ) revealed that all three chains have homologous pentapeptidyl sequences in the neighborhood of t w o regularly spaced cysteine residues [ 1 0 ] , raising the possibility that the three polypeptides have descended from a c o m m o n ancestor. Although we have commented on the structure-function consequences of fibrinogen evolving from a set of equivalent chains [ 11 ], we were frankly prepared for the eventuality that easily perceived homologies among the fibrinogen chains might have been blurred b y excessive amino acid replacements during the course of vertebrate evolution. Now, however, it is our contention that the occurrence of five identities in a string of six residues near the carboxy-terminus strongly favors the notion o f j3- and 7-chains arising as the result of gene duplication (cf. ref. 12). We are n o t y e t in a position to judge whether or n o t this was a discrete duplication leading to separate chains, or a contiguous (tandem) duplication giving rise to a single-chained profibrinogen [ 11 ]. ' B e y o n d the significance of c o m m o n ancestry for the j3- and 7-chains, a number of other interesting features have been revealed b y this investigation. The fact that the carboxy-terminus of the ~-chain remains intact during plasmin digestion under the conditions described, whereas the carboxy-terminus of the 7-chain is subject to plasmin attack, is at variance with the digestive scheme as described in some previous reports [13,14] b u t supportive of others [ 1 5 ] . In this regard, the apparent molecular weight of the unreduced fragment D used in our studies was 85 000 as determined on sodium dodecyl
620
Ly~-~e--~rg--Pro--Phe--Phe--Pro--Gln--Glnl CH-2
t
•'
CH-1
CN-9
"l "I
~ • - • IMet--LYs--IlelArgl Pr°--Phe - Phe--Pro--Gln--GlnC ~f'" '1Met--Lys--Ile~Ile--IPro--Phe" -Asn--Arg--Leu--Thr--Ile--Gly--Glu--Gly -28
-26
-22
-24
-20
-18
-16
Plasmin
l
•
--Gln--Gln--His--His--Leu~G1F--Gly--Ala~Lys--Gln--Ala--Gly--Asp--V ale -14
-12
-10
-8
-6
-4
-2
Fig.3. A m i n o a c i d s e q u e n c e s o f t h e c a r b o x y - t e r m i n a l r e g i o n s o f h u m a n f i b r i n o g e n ~- a n d 7 - c h a i n s . T h e data used to establish the sequence of the carboxy-terminal cyanogen bromide fragment of the ~-chain are summarized in the upper left hand comer: CN = cyanogen bromide; Ch = chymotrypsin; = carboxypeptidue A; =. ffi d e t e r m i n a t i o n b y t h e D N S - P I T C p r o c e d u r e . 7 - c h a i n r e s i d u e s are numbered negatively from the carboxy-terminus. Homologous regions are boxed. The * marks t h e g l u t a m i n e c r o s s l i n k i n g a c c e p t e r site; a b o n d a t t a c k e d b y p l u m t n d u r i n g t h e f o r m a t i o n o f f r a g m e n t D is n o t e d n e x t t o t h e l y s i n e c r o s s l i n k i n g d o n o r site.
sulphate polyacrylamide gel electrophoresis; gels run under reducing conditions yielded apparent molecular weights for the a-, ~- and 7-chain portions of 10 000, 44 000 and 35 000 respectively. It is also of interest to note that the removal of the pentapeptidyl sequence from the carboxy-terminus of the 7-chain ought to render these D fragments incapable of 7 ~ dimer formation by factor XIII, since the lysine donor residue is now the carboxy-terminal amino acid. On the other hand, the fragment still ought to be able to incorporate substitute donors since the glutamine accepter residue has not been violated [3]. Finally, some comment ought to be made regarding a previous report that the carboxy-terminal residue of the human fibrinogen ~-chain is valine [ 2 ] The low yields reported in that study raise the possibility that the ~-chains used may have been contaminated with 7-chains, the carboxy-terminals of
2.3 (2) .
Alanine
27
1.0 (1)
22
0.8 (1)
1.4 (2) .
.
2.3 (2) .
.
. 1.4 (3) 1 . 9 (2)
0.7 (1) .
3 . 2 (3) 1.1 ( I ) 4.0 (4)
1 . 0 (1) 2 . 2 (2)
.
.
.
1 . 0 (1) 1 . 8 (2)
.
0.9 (I) 2.6 (3) 2.1 ( 2 )
(4) (I) (5)
1,3 (I) 1.0 (I)
.
.
CN-IIIb6 ( F r a g m e n t D)
.
.
.
9
0.8 (1)
1.0 (1)
1.8 (2) .
. 0 . 7 (1) .
.
. 2.2 (2) 2.4 (2) .
. .
.
.
.
.
CN-IX (~-Chain)
.
.
.
. .
9
0.9 (1)
1.0 (1) .
2 . 2 (2)
.
. 0.8 (1)
. .
. 2 . 2 (2) 1.9 (2)
.
.
.
. .
.
CN-IVb-2 ( F r a g m e n t D)
.
.
.
.
.
. .
I S O L A T E D F R O M H U M A N F I B R I N O G E N ~- A N D
4
--
--
0 , 7 (1)
--
2 . 3 (2) 1 . 0 (1)
~-CN-IX-CH-1
5
0.9
(1)
1.2 (1)
1 . 0 (1)
0 . 6 (1)
-1.3 ( 1 )
~-CN-IX-CH-2
Chymotryptic peptides
CYANOGEN BROMIDE FRAGMENTS
* V a l u e s are given in r e s i d u e s p e r m o l . S a m p l e s w e r e h y d r o l y z e d f o r 2 4 h in 5.7 M HCI a t I I 0 ° C a n d t h e n a n a l y z e d o n a S p i n e o A u t o m a t i c A m i n o A c i d A n a l y z e r e m p l o y i n g a t h r e e - b u f f e r single c o l u m n p r o c e d u r e , All p e p t i d e s w e r e t r y p t o p h a n - n e g a t i v e . * * I n t h e case o f f r a g m e n t 7 C N - I V t h e v a l u e s are t h e a v e r a g e o f 2 4 a n d 4 8 h h y d r o l y s e s . T h e n e - n e b o n d in t h e s e p e p t i d e 8 i8 pa~ticttla~|y t e s / s t a n t t o a c i d h y d r o l y s i s , a n d we h a v e p r e v i o u s l y s h o w n t h a t 7 2 h h y d r o l y s i s is r e q u i r e d f o r q u a n t i t a t i v e r e c o v e r y [ 4 ] . T h i s a c c o u n t s f o r t h e p o o r ~ecoverY o f Isole1~cine i n t h e 2 4 h h y d r o l y s i s o f C N - I I I b 6 o b t a i n e d f r o m f r a g m e n t D.
Total residues
Cysteine V-line Isoleueine** Leucine Tyrosine Phenylalanine Histidine Lysine Homoserine Arginine
2.2 I.I . 4.2 0.9 4.9
Aspartic acid Threonine Serine Glutamic acid Proline Glyeine
(2) (1)
CN-IV** (T-Chain)
Amino acid
AMINO ACID COMPOSITIONS OF CARBOXY-TERMINAL 7 - C H A I N S A N D F R O M F R A G M E N T D*
TABLE I
O~ b0
622
which are undisputably valine [3]. Beyond that, the difficulties of determining carboxy-terminal glutamine residues are great. Not only does hydrazinolysis not release a free amino acid, but the free glutamine residue released by carboxypeptidase A is prone to cyclize into the elusive pyrrolidone carboxylic acid. There is always the alternative possibility, of course, that our ~-chains are the product of some premature proteolysis, but the excellent yields achieved in all experiments tend to disallow such an explanation. In summary, amino acid sequence studies on some key peptides isolated from human fibrinogen fragment D have provided valuable information about the structure and evolution of fibrinogen as well as shedding light on the sequence of events which occurs during the plasmic degradation of that molecule. The excellent technical assistance of Barbara Cottrell, Jay Solnick and Marcia Riley is gratefully noted. These studies were supported by N.I.H. Grants HE-12,759 and GM-17,702. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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