Open AccessPhotoaffinity labeling of the primary fibrin polymerization site: localization of the label to gamma-chain Tyr-363.
Author(s) -
Kensuke Yamazumi,
Russell F. Doolittle
Publication year - 1992
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.7.2893
Subject(s) - chemistry , peptide , alkylation , reagent , photoaffinity labeling , protein primary structure , amide , polymerization , fibrinogen , fibrin , chromatography , amino acid , biochemistry , peptide sequence , stereochemistry , binding site , organic chemistry , biology , polymer , immunology , gene , catalysis
Fragment D prepared from human fibrinogen was labeled specifically by photoactivation of the peptide [14C]Gly-Pro-Arg-N-(4-azido-2-nitrophenyl)Lys amide. The preparation was freed of excess labeling reagents and then reduced and alkylated. The component alpha, beta, and gamma chains were purified by chromatography on carboxymethylcellulose and the radioactivity was found to be restricted to the gamma chain. Isolated gamma chains were digested with various endopeptidases, both alone and in tandem, and the products were fractionated by gradient HPLC. The amino acid compositions of all labeled peptides led to the conclusion that the modification occurs exclusively on gamma-chain Tyr-363.