Purification and characterization of porcine C3. Studies of the biologically active protein and its split products.
Separation techniques for obtaining pure and biologically active swine C3 have been improved in this study. Using these procedures and through the further characterization of porcine C3, the possibilities for developing more specific techniques for the analysis of the complement system in swine have been improved. Plasma was initially treated with protease inhibitors, polyethylene glycol (PEG)-fractionation, plasminogen-depletion and a rapid chromatographic desalting step. The essential fractionation was carried out by DEAE-Sephacel chromatography. Contaminants were removed by size-exclusion (Sepharose CL-6B)- and hydroxylapatite-chromatography. The final recovery reached 56% with 73% retaining specific hemolytic activity. The amino acid composition (98.33%), the functional compatibility and the secondary structure of fragments and intact protein indicate a high degree of homology with human C3. In contrast with the findings of earlier studies was the considerable immunologic cross-reactivity observed with human C3, and the size difference between the human and the swine C3-beta subunit, which was found to be 10 kDa lighter than the human analogue. The finding that the swine C3b/iC3b/C3c fragments do not separate from C3 by agarose electrophoresis, unlike the human analogues, demonstrated that this commonly used simple parameter for the detection of complement activation cannot be used in the porcine model.
Storm
K E
KE
Department of Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden.
Arturson
G
G
Nilsson
U R
UR
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel / Q000662:veterinary; D002852:Chromatography, Ion Exchange / Q000662:veterinary; D003176:Complement C3 / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D003429:Cross Reactions / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D006801:Humans; D005779:Immunodiffusion / Q000662:veterinary; D007122:Immunoelectrophoresis / Q000662:veterinary; D013552:Swine / Q000276:immunology*
D000596:Amino Acids; D002241:Carbohydrates; D003176:Complement C3