Purification of protein C from canine plasmaReport as inadecuate




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BMC Veterinary Research

, 10:251

Clinical pathology, physiology and immunology

Abstract

BackgroundIn 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.

ResultsThe 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 O-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.

ConclusionsThese 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.

KeywordsDog Plasma protein Anticoagulant AbbreviationsFVFactor V

FVIIIFactor VIII

FDAFood and Drug Administration

EMEAEuropean Medicines Agency

PROWESSRecombinant human activated protein C worldwide evaluation in severe sepsis

CnPCCanine protein C

CnAPCCanine activated protein C

SDS-PAGESodium dodecyl sulphate polyacrylamide gel electrophoresis

DELTA-BLASTDomain-enhanced lookup time-accelerated BLAST

LC-MS-MSLiquid chromatography followed by tandem mass spectral analysis

IGOTIsotope-coded glycosylation site-specific tagging

PTMPost-translational modification

PVDFPolyvinylidene fluoride

PBS-TPhosphate-buffered saline, pH 7.4 containing 0.1% Tween-20

SEMStandard error of mean

APTTActivated partial thromboplastin time

Electronic supplementary materialThe online version of this article doi:10.1186-s12917-014-0251-2 contains supplementary material, which is available to authorized users.

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Author: Valerie M Wong - Dorothee Bienzle - M Anthony Hayes - Paul Taylor - R Darren Wood

Source: https://link.springer.com/



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