Fluorescence resonance energy transfer study of the associative state of membrane-bound complexes of complement proteins C5b-8.
Human complement protein C8 was labeled with the fluorescent chromophores fluorescein-5-isothiocyanate (FITC), 3-(4-isothiocyanatophenyl)-7-diethylamine-4-methyl coumarin (IPM), eosin-5-isothiocyanate (EOS), or Texas Red (sulforhodamine-101-sulfonyl chloride; TR) with only minor reduction in the specific hemolytic activity of the protein. The distribution of C5b-8 complexes bound to sheep erythrocyte membranes was investigated by monitoring fluorescence resonance energy transfer (RET) between the following RET donor/acceptor pairs of labeled C8: FITC-C8/EOS-C8, IPM-C8/EOS-C8, and FITC-C8/TR-C8. On binding to membranes containing pre-formed C5b67 complexes, specific RET was detected for each of the donor/acceptor pairs of labeled C8 investigated. In contrast, no energy transfer was observed for these RET donor/acceptor pairs of labeled C8 incubated in the presence of control membranes or in membrane-free solution. On the basis of a consideration of the transfer efficiency that would be expected for donor/acceptor pairs of labeled C8 that were uniformly dispersed on the membrane surface, these results suggest that C5b-8 complexes are aggregated into polymeric clusters when membrane-bound. The efficiency of donor-C8 to acceptor-C8 RET--and the hemolytic activity of membrane-bound C5b-8 (in the absence of C9)--are both related to the surface density of membrane-bound C5b67, suggesting that the physical clustering of the membrane-inserted C5b-8 complex may be related to the expression of its cytolytic activity.
D002463:Cell Membrane Permeability; D003167:Complement Activation; D003185:Complement C8 / Q000378:metabolism*; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins / Q000378:metabolism*; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates; D013997:Time Factors
D003185:Complement C8; D015938:Complement Membrane Attack Complex; D005452:Fluoresceins; D013861:Thiocyanates; D003165:Complement System Proteins; D016650:Fluorescein-5-isothiocyanate