On the Primary Structure of Human Fibrinogen. Isolation and Characterization of N-Terminal Fragments from Plasmic Digests

Abstract

Two N-terminal fragments of a(A)-chain and /l(B)-chain in human fibrinogen have been isolated from a plasmic hydrolyzate. The fragment from the u(A)-chain consisted of 43 amino acid residues including two half-cystine residues. On treatment with thrombin, this fragment produced two other peptides in addition to fibrinopeptideA and its analogues. One was a tripeptide, Gly-Pro-Arg, and the other a peptide containing 24 amino acid residues having N-terminal valine. The partial amino acid sequence of the a(A)-chain has been found to be: Ala-AspSer-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-~g-His-G~-Ser-Ala-Cys-Lys-Asp-Ser-Asp-Trp-Pro-Phe-(Cys-Ser-Asp-Glu-Trp-Asn-Tyr)-Lys. I IThe fragment from the /?(B)-chain consisted of 21 amino acid residues. This fragment released fibrinopeptide B on treatment with thrombin. The amino acid sequence of the B(B)-chain fragment is: Pyr-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-~g-Pro-Leu-Asp-Lys. The linkages between fibrinopeptides and fibrin, which are rapidly hydrolyzed by thrombin, are very resistant towards plasmin.A number of studies on mammalian fibrinogen and fibrin have suggested that both protein molecules are made up of three polypeptide chains, u("A"), /?(B) and y [l--51, respectively. The N-terminal parts of the u("A'))-chain and /?(B)-chain of fibrinogen contain fibrinopeptides "A" and B, which are split off by thrombin during the fibrinogen-fibrin transition Thrombin has a very limited proteolytic action on fibrinogen, resulting in the release of fibrinopeptide "A" and B [6-101. There is strong evidence that arginyl-glycyl peptide bonds in the N-terminal parts of the a("A")-chain and /l(B)-chain in fibrinogen are split by thrombin [S-10,291. Plasmin, which like thrombin also is a proteolytic enzyme with trypsinlike specificity, does not liberate fibrinopeptide A and B from fibrinogen or S-sulfo-fibrinogen [30,31], indicating that the bonds split by thrombin are resistant towards plasmin. One would therefore expect to find polypeptide fragments containing covalently linked fibrino-peptides in plasmic digests of fibrinogen. Isolation and structural studies on such fragments is the subject of this paper. A preliminary note on this work has appeared [31]. MATERIALSHuman fibrinogen waa prepared from pooled citrated plasma according to the method of Blomback and Blombiick [32]. The coagulability of the preparation, as determined spectrophotometrically[33], was 97-100 Ole.

Preparation and isolation of the S‐carboxymethyl derivative chains of human fibrinogen

“…Fibrinopeptides A and B are cleaved off the a(A) and P(B) chains respectively by thrombin. The resulting product is fibrin [3,4]. Sulfitolysis of fibrinogen results in the cleavage of the disulfide bridges [5] , and the resulting S-sulfo chain derivatives have been separated by paper [ 51, starch gel electrophoresis [6] and column chromatography [S, 71. More recently, the S-sulfo chain derivatives from bovine [8] and human [9] fibrinogen have been isolated on carboxymethyl cellulose using a stepwise [8] and gradient [9] sodium acetate buffer system.…”

Primary structure of the 16S rRNA binding protein S15 from Escherichia coli ribosomes

“…Cyanogen bromide cleavage of the protein S15 was performed in 70% formic acid at room temperature for 20 h and the resulting peptides were separated by gel filtration on Biogel P-30 in a 5% acetic acid solution. Amino acid compositions of the peptides were determined with an automatic amino acid analyzer (JEOL; type JLC6AH) after hydrolysis with 5.7 N HCl containing 0.02% mercaptoethanol at 108°C for 20 h. The amino acid sequence of the peptides was determined by manual Edman degradation as modified by Iwanaga et al [7] and the PTH-amino acids were identified by thin-layer chromatography on silica gel plates.…”

Amino acid sequence of cardiotoxin from the venom of Naja naja atra

“…Amino acid analyses of cardiotoxin and the peptides generated by cyanogen bromide cleavage or tryptic digestion of the RCM-toxin were performed with the standard method using an Hitachi KLA 3B amino acid analyzer. Sequence analyses were conducted by the modified Edman procedure [19,20], and the phenylthiohydantoin amino acids were identified by thin layer chromatography employing several solvent systems [21][22][23]. Carboxypeptidase A digestion was performed according to standard methods [24].…”