The Complex of C5b and C6: Isolation, Characterization, and Identification of a Modified form of C5b Consisting of Three Polypeptide Chains

Abstract

A new method for isolation of the complex of C5b and C6 (C5b-6) which yielded milligram quantities of C5b-6 of high purity was devised. C5 and C6 first were separated from C7 by column chromatography on DEAE-cellulose. C5b-6 then was generated from the C7-free reagent by incubation with serum-treated zymosan, and purified by DEAE-cellulose and Bio-Gel A 1.5 m column chromatography. Upon analysis by sucrose density gradient ultracentrifugation and gel filtration, two forms of the C5b-6 complex were recognized. The first maintained its complexed state (10.4S) during ultracentrifugation, and formed aggregates (>16S) upon reaction with C7. The second and predominant form dissociated into C5b (7.6S) and C6 (5.7S) during ultracentrifugation and did not form aggregates with C7. Both forms retained their elution characteristics on gel filtration over a 1-month period at 4°C. SDS-polyacrylamide gel electrophoretic analysis also revealed two different forms of the C5b-6 complex. C5b-6 capable of aggregating with C7 was a complex of unmodified C5b and C6. The predominant form was, however, a complex of modified C5b (designated C5b′) and C6. In the absence of a reducing agent, both forms of C5b had an apparent m.w. of 180,000 daltons. Upon reduction, C5b yielded two polypeptide chains designated α′ (103,000 daltons) and β (75,000 daltons), whereas C5b′ dissociated into three polypeptide chains, i.e., two fragments of the α′ chain designated α′1 (75,000 daltons) and α′2 (27,500 daltons), and the β chain (75,000 daltons). The possible significance of this proteolytic modification of C5b in relation to the hemolytic efficiency of lysis mediated by C5b-6 is discussed.