Kinetics of polymerization of a fluoresceinated derivative of complement protein C9 by the membrane-bound complex of complement proteins C5b-8

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Abstract
The fluorescence self-quenching by energy transfer of FITC-C9 [fluorescein isothiocyanate-complement component 9], a fluoresceinated derivative of human complement protein C9 (Sims, P.J., 1984), was used to monitor the kinetics of C9 polymerization induced by the membrane-associated complex of complement proteins C5b-8. Time-based measurements of the fluorescence change observed during incubation of FITC-C9 with C5b-8-treated sheep red blood cell ghost membranes at various temperatures revealed that C9 polymerization induced by the C5b-8 proteins exhibits a temperature dependence similar to that previously reported for the complement-mediated hemolysis of these cells, with an Arrhenius activation energy for FITC-C9 polymerization of 13.3 .+-. 3.2 kcal mol-1 (mean .+-. 2 SD). Similar measurements obtained with C5b-8-treated unilamellar vesicles composed of either egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine or dimyristoylphosphatidylcholine revealed activation energies of between 20 and 25 kcal mol-1 for FITC-C9 polymerization by C5b-8 bound to these membranes. Temperature-dependent rates of C9 polymerization were observed to be largely unaffected by the phase state of membrane lipid in the target C5b-8 vesicles. The significance of these observations to the mechanism of C9 activation of membrane insertion is considered.

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