Mode of Action of Human C9: Adsorption of Multiple C9 Molecules to Cell-Bound C8

Abstract

The following experimental observations were made: 1) The C9 hemolytic dose response curve is sigmoidal; 2) C9 binding to EAC1-8 at low C9 input shows a definite time lag; 3) binding of three to six C9 molecules is required for the formation of a functional lesion (63% lysis); 4) C9 binding is relatively insensitive to temperatures from 0° to 43°C, whereas hemolysis of EAC1-9 is highly temperature sensitive; 5) C9 binding is independent of pH between 6.0 and 8.5 and of ionic strength over the range µ = 0.05 to µ = 0.15; and 6) the removal of C9 hemolytic activity from the fluid phase by serial adsorption with EAC1-8 is fully accounted for by the physical uptake of C9 molecules to EAC1-8. The results indicate that expression of C9 hemolytic activity requires physical binding of C9, that binding is achieved by adsorption through a cooperative process, and that binding of more than one molecule of human C9 is necessary for the formation of one functional membrane lesion. The manifestation of functional lesions is dependent on temperature allowing membrane fluidity.