Identification of disulfide-bridged substructures within human von Willebrand factor.
In the course of identifying substructural domains within the homooligomeric protein von Willebrand factor [270 kilodaltons (kDa) per polypeptide chain], seven large fragments of 8-90 kDa have been generated by limited proteolysis. A monomeric fragment that binds coagulation factor VIIIc is identified as residues 1-272. A fragment that binds platelet glycoprotein Ib is identified as a homodimer containing two pairs of identical chains, i.e., residues 273-511 and 674-728. Disulfide bonds have been identified by several methods, including direct observation of the phenylthiohydantoin of cystine during Edman degradation of isolated peptides. Among half-cystine residues in the amino-terminal 1365-residue region, 52 have been paired. They place structural constraints on folding possibilities within three structural domains. Additional clusters of disulfide bonds are evident. It has been shown that at least 35 disulfides must form intrachain bridges, specifically the cystines among residues 1-272 and 906-1492. Intersubunit disulfide bonds are partially localized in an interior region (residues 283-695) and a carboxyl-terminal region (residues 1908-2050). Each of these regions appears to be linked to a corresponding region of a neighboring subunit in the network of interconnected chains. The difficulties of pairing all 169 half-cystines (per chain) and of distinguishing intrachain from interchain disulfides are evaluated.
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D013820:Thermolysin; D014841:von Willebrand Factor / Q000378:metabolism
D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014841:von Willebrand Factor; D013820:Thermolysin; D003488:Cyanogen Bromide