title: Isolation and partial structural characterization of an equine fibrinogen CNBr fragment that exhibits immunologic cross-reactivity with an A alpha-chain cross-linking region of human fibrinogen.
abstract: Immunochemical studies of equine fibrinogen were conducted to characterize the structural basis for the immunologic cross-reactivity observed between human and equine A alpha chains when employing an antiserum to the 26K, human cyanogen bromide (CNBr) fragment, A alpha 241-476 (CNBr VIII). A 38K, equine CNBr fragment that reacts with this antiserum was isolated from CNBr-digested equine fibrinogen by Sephadex G-100 gel filtration. It was further purified by sequential hydrophobic chromatography on phenyl-Sepharose CL-4B, followed by reversed-phased (C-8) high-performance liquid chromatography (HPLC). NH2-Terminal analysis of the purified fragment, designated EqA alpha CNBr, identified one major sequence whose first three residues, E-L-E, were identical with those of human CNBr VIII. Tryptic and staphylococcal protease digests of the equine fragment were resolved by reversed-phase HPLC (C-4, C-18), and the separated components were characterized by amino acid analysis and automated Edman degradation. A total of 34 tryptic and 20 staph protease peptides yielded sequence information that permitted the alignment of 271 equine residues with residues A alpha 241-517 from the COOH-terminal two-thirds of the human A alpha chain so that 63% of the possible matches were identical. Other features of interest included (1) an amino acid substitution in which the methionine residue at A alpha 476 in the human A alpha chain was replaced by a valine residue, thus accounting, in part, for the larger EqA alpha CNBr fragment obtained from the equine molecule, and (2) a region of striking homology in which 36 successive residues, corresponding to A alpha 428-464 in the human A alpha chain, were identical in both species. These findings, together with available structural data for the COOH-terminal portion of the rat and bovine A alpha chains, indicate that the region corresponding to (human) A alpha 240-517 represents a conserved portion of the fibrinogen molecule. This may, in turn, explain the difficulties encountered when trying to raise monoclonal antibodies to cross-linking regions that are contained within the COOH-terminal two-thirds of the human A alpha chain.
authors: Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032.
authors: J H
authors: JH
authors: Sobel
authors: C A
authors: CA
authors: Thibodeau
authors: M A
authors: MA
authors: Kolks
authors: R E
authors: RE
authors: Canfield
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D005340:Fibrinogen / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
chemical_list: D003432:Cross-Linking Reagents; D005340:Fibrinogen; D003488:Cyanogen Bromide