DOMAIN STRUCTURE OF PROTEIN C
The domain structure of protein C is represented, where: GLA = region containing γ-carboxyglutamic acid residues, EGF = region containing sequences homologous to human epidermal growth factor, AP = activation peptide released upon conversion of the zymogen to the active serine protease, CATALYTIC DOMAIN = region containing the serine protease catalytic triad. The arrow indicates the site which is proteolytically cleaved by thrombin during activation of the zymogen.
|Plasma concentration:||4-5 µg/ml (human)
5-10 µg/ml (bovine)
|Mode of action:||Zymogen; precursor to the serine protease activated protein C (APC)|
|Molecular weight:||62,000 (human)
|Isoelectric point:||4.4-4.8 (human)
|Structure:||two chains, Mr=41,000 and 21,000, disulfide linked, NH2-terminal gla domain two EGF domains|
|Percent carbohydrate:||23 % (human)
14 % (bovine)
|Post-translational modifications:||eleven gla residues (bovine), nine gla residues (human), one β-hydroxyaspartate|
50% (vol/vol) glycerol/H2O
>95% by SDS-PAGE
NOT tissue/cell culture grade. Not tested for endotoxin.
< 0.5% aPC activity by chromogenic assay
Shelf Life (properly stored)
Gel: Novex 4-12% Bis-Tris
Load: Human Protein C, 1 µg per lane
Standard: SeeBluePlus 2; Myosin (191 kDa), Phosphorylase B (97 kDa), BSA (64 kDa), Glutamic Dehydrogenase (51 kDa), Alcohol Dehydrogenase (39 kDa), Carbonic Anhydrase (28 kDa), Myoglobin Red (19 kDa), Lysozyme (14 kDa)
The vitamin K-dependent zymogen, protein C, is synthesized in the liver as a single chain polypeptide and is subsequently converted to a disulfide linked heterodimer, by removal of a dipeptide (Lys-146 and Arg-147) from the precursor molecule. Trace quantities of the single chain form have been observed in plasma. The light chain, which is responsible for the calcium dependent binding of protein C to phospholipid vesicles, contains 11 γ-carboxyglutamic acid (gla) residues, 1 b-hydroxyaspartic acid residue, and 2 epidermal growth factor (EGF) homology domains. The serine protease catalytic triad is located in the heavy chain. Human protein C is susceptible to proteolytic cleavage of a peptide (Mr=3000) from the COOH-terminal end of the heavy chain, yielding an altered form referred to as β-protein C. No functional distinction between α- and β-protein C has been observed. A single cleavage at Arg-12 (Arg-14 in bovine) of the heavy chain of human protein C converts the zymogen into the serine protease, activated protein C. This cleavage is catalyzed by a complex between α-thrombin and the endothelial cell surface protein thrombomodulin. In contrast to the other vitamin K dependent coagulation factors, activated protein C functions as an anticoagulant by catalyzing the proteolytic inactivation of factors Va and VIIIa. APC also contributes to the fibrinolytic response by complex formation with plasminogen activator inhibitors.
Bovine protein C is prepared from fresh citrated bovine plasma by a modification of the Walker procedure, as described by Haley et al. Human protein C is prepared from fresh frozen citrated human plasma using a combination of immunoaffinity chromatography, and conventional techniques. Protein C is provided in 50% (vol/vol) glycerol/H2O and should be stored at -20°C. Purity is determined by SDS-PAGE analysis and activity is measured using a chromogenic substrate based assay.