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Human C3b Inactivator (C3b INA): A New Purification Method, and Studies with the Highly Purified Protein

Department of Medicine, University of Miami School of Medicine, Miami, Fla.

Abstract

C3b INA was purified from normal human serum by a five-step method at 4°C. The method separates C3b INA from C2, C6, C7, C8, IgG, IgA, hemopexin, and transferrin, some of which have presented problems in other reported procedures. Activity was tested with EAC43 by the immune-adherence inhibition assay. Step 1: C1 was precipitated at low ionic strength, pH 7.5 Step 2: The supernatant was applied to a DEAE-cellulose column (buffer; 5 mM sodium phosphate, 70 mM NaCl, 2 mM EDTA, pH 7.5). All of the C3b INA was found in an effluent protein peak. Step 3: Fifteen per cent polyethylene glycol "6000" and 10 mM EDTA (protein adjusted to 3 mg/ml) precipitated most of the C8, IgG, and IgA, whereas most of the transferrin, hemopexin, C2, and C3b INA were in the supernatant. Step 4: Hydroxylapatite chromatography (buffer; 50 mM Na, K phosphate, pH 6.9). A linear gradient of starting buffer and 300 mM K phosphate caused the elution of C3b INA at a conductance of 10 mmhos/cm, whereas C2 eluted later.