Purification of C3b Inactivator and Demonstration of Its Two Polypeptide Chain Structure

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Abstract
C3b inactivator (C3bINA) was isolated from plasma by sequential ammonium sulfate gradient solubilization, anion exchange chromatography, gel filtration, and repeat ammonium sulfate gradient solubilization. The final product was pure as assessed by alkaline disc gel electrophoresis, isoelectric focusing, and SDS polyacrylamide gel electrophoresis, and elicited a monospecific rabbit antiserum. The normal serum concentration of C3bINA was found to be 53 ± 9 µg/ml (mean ± S.D.). Heterogeneity of purified C3bINA was apparent on alkaline disc gel electrophoresis and isoelectric focusing, but not with SDS polyacrylamide gel electrophoresis and thus is attributed to forms of C3bINA that differ in charge rather than in size. SDS polyacrylamide gel electrophoresis of unreduced, alkylated C3bINA yielded a single stained band with an apparent m.w. of 93,000, whereas the reduced protein demonstrated two bands of 55,000 and 42,000 m.w., thereby establishing a composition of two disulfidelinked polypeptide chains for C3bINA.