Amino acid sequence studies on the alpha chain of human fibrinogen. Exact location of cross-linking acceptor sites.
Human fibrinogen was clotted under conditions that promote latent factor XIII activity and in the presence of a radioactive substitute cross-linking donor ([14C]glycine ethyl ester). The labeled fibrin was reduced and alkylated in the presence of 6 M guanidinium chloride. After dialysis and freeze-drying, the preparation was separated into its constituent polypeptide subunits by chromatography on (carboxymethyl)cellulose in the presence of 8 M urea. Under the incorporation conditions used, the radioactivity was limited to gamma chains (one donor molecule/chain) and alpha chains (two donor molecules/chain). The labeled alpha chains were digested with cyanogen bromide and fractionated on Sephadex G-50. All the radioactivity was found in a fragment previously designated H alpha CNI, the largest of the cyanogen bromide fragments in the alpha chain. The fragment was further fragmented by digestion with plasmin, trypsin, chymotrypsin, and/or staphylococcal protease. The incorporated radioactivity was found to reside in equal amounts at two different sites located 38 residues apart. These were determined to be positions 88 and 126 in H alpha CNI, which correspond to glutamine-328 and glutamine-366 in the alpha chain.
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D005176:Factor XIII / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006146:Guanidines; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases
D003432:Cross-Linking Reagents; D006146:Guanidines; D010446:Peptide Fragments; D005340:Fibrinogen; D005176:Factor XIII; D010447:Peptide Hydrolases