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Purification of the First Component (C'1) of Complement by Zonal Ultracentrifugation and of the Second (C'2) by Electrofocusing

National Cancer Institute, Bethesda, Md.

Abstract

The zonal centrifuge has been used to purify viruses, subcellular organelles and macromolecules of biologic interest. In this report we describe a preparative zonal ultracentrifugation method for the separation from serum of chemically and functionally highly purified first component (C'1) of guinea pig and human complement. The observation that the sedimentation rate of C'1 varies inversely with the ionic strength of the medium made the development of this purification procedure possible. At an ionic strength of 0.3 or greater the sedimentation constant of C'1 is about 4 and at an ionic strength of 0.15 or less the sedimentation constant of C'1 is about 18. The purification procedure consists of a two-cycle centrifugation: the first at µ = 0.75 separates C'1 from 19 S serum proteins; the cycle at µ = 0.065 separates C'1a, now an 18 S molecule, from the smaller molecules.