title: Purification of protein C from canine plasma.
abstract: BACKGROUND
  In order to characterize the functional properties of canine protein C (CnPC), the zymogen needs to be purified from plasma. The goals of this study were (1) to purify protein C from fresh frozen canine plasma by barium chloride and ammonium sulphate precipitation, followed by immunoaffinity chromatography using a monoclonal mouse antibody against human protein C (HPC4) and (2) to characterize this protein's structure.
  
  
  RESULTS
  The purified protein contained three glycosylated forms of a heavy chain (~49 kDa) and a glycosylated light chain (~ 25 kDa). Tandem mass spectra of the peptides obtained following trypsin digestion and liquid chromatography identified this protein to be protein C (vitamin K-dependent protein C precursor, gi|62078422) with 100% probability. Three glycosylation sites (Asn139, Asn202, and Asn350) were identified by detection of peptides containing an N-linked glycosylation consensus sequon with a 3-dalton increase in mass following incubation of the protein with PNGase F in 18O-labeled water. Following incubation with Protac (a specific activator of protein C), the heavy chain showed a slight decrease in molecular size and amidolytic activity measured by a synthetic chromogenic substrate containing an amide bond [H-D-(γ-carbobenzoxyl)-lysyl-prolyl-arginine-paranitroanilide diacetate salt]. The amidolytic activity was increased by ~303-fold in the final protein preparation compared to that in plasma. The purified protein showed concentration-dependent anti-factor V and anti-factor VIII activities in canine plasma in coagulometric factor assays.
  
  
  CONCLUSIONS
  These studies showed that CnPC could be purified from plasma using HPC4 and that this protein showed amidolytic and anti-coagulant properties upon activation with Protac.
authors.forename: Valerie M
authors.initials: VM
authors.lastname: Wong
authors.forename: Dorothee
authors.initials: D
authors.lastname: Bienzle
authors.forename: M Anthony
authors.initials: MA
authors.lastname: Hayes
authors.forename: Paul
authors.initials: P
authors.lastname: Taylor
authors.forename: R Darren
authors.initials: RD
authors.lastname: Wood
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004285:Dogs / Q000097:blood*; D010949:Plasma / Q000737:chemistry*; D011486:Protein C / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011486:Protein C