Purification of protein C from canine plasma.

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. 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. 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.

Citation

Valerie M Wong, Dorothee Bienzle, M Anthony Hayes, Paul Taylor, R Darren Wood. Purification of protein C from canine plasma. BMC veterinary research. 2014 Oct 18;10:251

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PMID: 25326145

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