Amino acid sequence studies on the .alpha. chain of human fibrinogen. Exact location of crosslinking acceptor sites

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Abstract
Human fibrinogen was clotted under conditions that promote latent factor [F] 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 (CM)cellulose in the presence of 8 M urea. Under the incorporation conditions used, radioactivity was limited to .gamma. chains (1 donor molecule/chain) and .alpha. chains (2 donor molecules/chain). 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 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 resided in equal amounts at 2 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.