Photoaffinity labeling of the primary fibrin polymerization site: isolation and characterization of a labeled cyanogen bromide fragment corresponding to gamma-chain residues 337-379.

Abstract

Human fibrinogen and the plasmin-generated fibrinogen fragment D were photoaffinity labeled specifically with the peptide [14C]Gly-Pro-Arg-N(4-azido-2-nitrophenyl)Lys amide. In the case of fibrinogen, greater than 85% of the incorporated radioactivity was found in the gamma chain. Similarly, when fragment D (Mr, 90,000) was labeled with the same derivatized peptide, virtually all the radioactivity was found in the gamma-chain portion. The labeled fragment D was treated with CNBr and an initial purification was achieved by two gel-filtration steps. The labeled material was purified further by HPLC and was also compared with CNBr digests of unlabeled material. Amino acid analysis and gas-phase sequencing showed the labeled fragment to be gamma-chain residues 337-379.