Open AccessLocalization of the cross‐linking site of GPRVVERHK in the γ‐chain of human fibrinogen
Author(s) -
CIERNIEWSKI Czeslaw S.,
BUDZYNSKI Andrei Z.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18380.x
Subject(s) - plasmin , chemistry , peptide , fibrinogen , peptide sequence , fibrin , biochemistry , binding site , chromatography , microbiology and biotechnology , enzyme , biology , immunology , gene
The peptide α17–24‐Lys (GPRVVERHK) corresponding to the N‐terminus of the α chain of fibrin was synthesized and used to localize its binding site in the fibrinogen molecule. The peptide was radioiodinated, incubated with fibrinogen, cross‐linked with a bifunctional reagent disuccinimidyl suberate and the resulting product was analyzed in several ways, including plasmin digestion. The binding of the radioactive peptide was mainly to the γ‐chain and was inhibited by unlabelled GPRVVERHK and GPRP. After plasmin digestion, the radioactivity was present in fragment D 1 and also in its γ‐chain remnant, but not in fragments D 2 , D 3 or E 3 . Fragment D 1 cross‐linked with iodinated GPRVVERHK was purified by affinity chromatography on immobilized anti‐fragment D IgG, further digested with plasmin in the presence of EGTA and the peptides were fractionated by reverse‐phase HPLC. The amino acid sequence analysis of the radioactive peak revealed the presence of two peptides, γ357–373 and GPRVVERHK. It was concluded that the binding site for GPRVVERHK is in the sequence γ357–373 which is present in fragment D 1 but absent in fragments D 2 and D 3 .