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 γ chains (one donor molecule/chain) and α chains (two donor molecules/chain). The labeled α chains were digested with cyanogen bromide and fractionated on Sephadex G-50. All the radioactivity was found in a fragment previously designated HaCNI, the largest of the cyanogen bromide fragments in the α 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 HαCNI, which correspond to glutamine-328 and glutamine-366 in the α chain. © 1979, American Chemical Society. All rights reserved.