Purification and physicochemical properties of C1q from guinea-pig serum |
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Authors: | T Hitschold M D Golan U Rabs M Loos |
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Affiliation: | Institut für Medizinische Mikrobiologie, Johannes Gutenberg-Universität, D-6500 Mainz, F.R.G. |
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Abstract: | An efficient method is described for the isolation of highly purified, IgG-free and stable guinea-pig serum C1q. The procedure includes the chromatography of EDTA-treated serum (25 mM EDTA) on CM- and DEAE-cellulose followed by gel filtration on ACA 34-Ultrogel whereby ammonium sulfate precipitation was used for concentration. The final product stored in a glycerol containing buffer was purified 700-fold with a yield of approximately 50%. It was judged to be homogeneous by several criteria including SDS-PAGE, analytical ultracentrifugation, gel filtration and immunoprecipitation. The protein has a sedimentation rate of 11.3 S and consists of three distinct polypeptide chains A, B and C with mol. wts of 30,200, 28,200 and 24,000. Amino acid analysis revealed a content of 4.42% hydroxyproline, 1.81% hydroxylysine and 18.7% glycine. In contrast to human serum C1q a very low content of cysteine residues was detected. SDS-PAGE analysis performed in the absence of 2-mercaptoethanol but in the presence of 5-10% SDS revealed clearly that gps-C1q is dissociated in a time-dependent manner into the individual chains. |
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Keywords: | Sheep erythrocytes (E) sensitized with rabbit IgG antibody (A) containing complement (C) components EAC1 EAC14 EDTA ethy-lenediaminetetraacetate VBS veronal-buffered saline VBS-S veronal-buffered saline with sucrose gps normal guinea pig serum ‘ACA-buffer’ pH 8.6 10% sucrose 20% glycerol SDS-PAGE sodium dodecyl sulfate-polyac-rylamide gel electrophoresis TCA 1,1,1-trichloroacetic acid 2-ME 2-mercaptoethanol |
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