title: Molecular characterization of antithrombin Barcelona-2: 47 arginine to cysteine.
abstract: The molecular characterization of antithrombin Barcelona-2 is reported. The abnormal antithrombin was isolated from plasma by chromatography on heparin-Sepharose at pH 6.0, and ion exchange on DEAE-Sephadex at pH 8.6 and 6.0. The tryptic peptides were mapped by reverse-phase HPLC and amino acid sequencing and mass spectrometry showed arginine-47 to be replaced by cysteine. The affinity of Barcelona-2 for heparin is dramatically decreased. The new cysteine does not form a mixed disulphide with DTNB, implying it is present as a disulphide with some other available thiol molecule such as cysteine. This extra bulk at position 47 accounts for the low heparin affinity compared with two other mutations (Rouen-1 47 His; Rouen-2 47 Ser) at this residue. These results confirm the view that Arg-47 is an important residue in heparin binding. No dimers of Barcelona-2 were observed suggesting that steric hindrance of the new cysteine at residue 47 prevents dimerisation.
authors.affiliation: Molecular Pathology Laboratory, Christchurch Hospital, New Zealand.
authors.forename: M C
authors.initials: MC
authors.lastname: Owen
authors.forename: G J
authors.initials: GJ
authors.lastname: Shaw
authors.forename: E
authors.initials: E
authors.lastname: Grau
authors.forename: J
authors.initials: J
authors.lastname: Fontcuberta
authors.forename: R W
authors.initials: RW
authors.lastname: Carrell
authors.forename: D R
authors.initials: DR
authors.lastname: Boswell
mesh_terms: D000595:Amino Acid Sequence; D058833:Antithrombin Proteins; D000991:Antithrombins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005343:Fibrinolytic Agents; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D013917:Thrombin / Q000378:metabolism
chemical_list: D058833:Antithrombin Proteins; D000991:Antithrombins; D005343:Fibrinolytic Agents; C061453:antithrombin Barcelona 2; D006493:Heparin; D013917:Thrombin