Soluble C5b-9 complex of guinea pig complement: Demonstration of its heterogeneity and the mechanism of its C9 hemolytic activity as transfer of reversibly bound C9 molecules from the complex

Abstract

Doubly radiolabeled soluble complex of the late acting components (SC5b-9) was formed by activating guinea pig complement (C)† serum mixed with C5 and C9 each labeled with either 125I or 131I with zymosan. It was isolated by sequential gel filtrations and sucrose density gradient centrifugation. Fractions of the complex obtained by centrifugation were heterogeneous in size, composition and hemolytic activity as C9. The heavier fractions contained four to six molecules of C9 per C5b molecule. The lighter fractions contained two to three C9 molecules to one C5b molecule and had stronger C9 hemolytic activity. On recentrifugation on the same sucrose density gradient, the factions moved to the same positions as on the first centrifugation. Guinea pig SC5b-9 complex bound to the surface of EA, EAC-3, EAC-5, EAC-6, EAC-7 and EAC-8 in similar amounts. Therefore, combination of the complex with these intermediate cells seems to be nonspecific. EAC-7 cells bound to the complex could be lysed by addition of C8. Lighter, hemolytically more active complex purified by recentrifugation was stable when incubated alone in buffer. But when it was incubated with free C9, it released radiolabeled C9, the amount released increasing with increase in the amount of free C9 added to a maximum of 10 to 15% of the C9 in the complex. A minute part of the complex was converted to heavier complex by incorporation of additional C9, but most of the complex did not change in size. Therefore, it did not seem to be an uncompleted complex to be saturated with more C9. The C9 molecules released from the complex were in an active form and could be reincorporatd into SC5b-9 when they were treated with zymosan in fresh guinea pig serum. It is concluded that the C9 hemolytic activity of guinea pig SC5b-9 is exerted by transfer of reversibly bound C9 molecules in the complex to C5b-8 sites on EAC-8 cells, not by the action of the complex in toto.