Isolation of the fourth component of guinea pig complement and its single polypeptide chain precursor from plasma |
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Authors: | Joseph Burge Anne Nicholson-Weller K.Frank Austen |
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Affiliation: | 1. Department of Medicine, Harvard Medical School, Department of Rheumatology and Immunology, Brigham and Women''s Hospital U.S.A.;2. Department of Medicine, Beth Israel Hospital, Boston, MA 02115, U.S.A. |
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Abstract: | Guinea pig C4 (C4gp)4 was isolated from plasma by polyethyleneglycol-4000 (PEG-4000) precipitation, lysine-Sepharose affinity chromatography removal of plasminogen and plasmin, DEAE-Sephacel, SP-Sephadex chromatography, and Sepharose CL-6B gel filtration. Both phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetraacetate (EDTA) were present in all Chromatographie buffers, and each C4 pooled concentrate was made 0.5 mM in DFP. The overall yield for three consecutive preparations averaged 3.1% of the initial plasma hemolytic C4 activity and the average specific activity rose to 139,223 U/mg, representing an average purification factor of 157-fold. The preparation of C4 gave a single band on Ouchterlony analysis with antiserum to guinea pig serum or to purified C4. Analytical alkaline discontinuous gel electrophoresis revealed a dominant protein band and functional C4 eluted from replicate gels in this region. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of unreduced C4 gave a mol. wt of 200,000, whereas SDS-PAGE of reduced and alkylated C4 demonstrated subunits of 93,000, 75,000 and 33,000 mol. wt as well as a small amount of non-reducible 200,000 mol. wt material. Elution of the 200,000 mol. wt material recognized by SDS-PAGE under reducing conditions revealed antigenic relatedness to either the α- or β-chain of C4, suggesting that it represents a pro-C4 single chain polypeptide. |
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Keywords: | fourth component of guinea pig complement DFP diisopropylfluorophosphate EAC1 sheep erythrocyte (E) bearing specific antibody (A) and the first component of complement (Cl) gp guinea pig EDTA ethylenediamine tetraacetate hu human HSA human serum albumin PMSF phenylmethylsulfonyl fluoride PEG-4000 polyethyleneglycol-4000 SDS sodium dodecyl sulfate PAGE polyacrylamide gel electrophoresis VBS veronal buffered saline mS milliSiemens |
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