Complement protein C9 labeled with fluorescein isothiocyanate can be used to monitor C9 polymerization and formation of the cytolytic membrane lesion.
Human complement protein C9 was covalently labeled with the fluorescent chromophore fluorescein isothiocyanate (FITC) with only a small reduction in the cytolytic activity of the protein. Polymerization of the labeled protein--either by incubating with lipid vesicles treated with complement proteins C5b-8 (activating the C5b-9 membrane lesion) or by heating the protein [Tschopp, J., Muller-Eberhard, H.J., & Podack, E.R. (1982) Nature (London) 298, 534]--resulted in a 40-60% decrease in the fluorescence emission from FITC. The decrease in total fluorescence was accompanied by an increase in the steady-state anisotropy following activation and polymerization of FITC-C9 by C5b-8 membranes, while heat-induced aggregation of the protein resulted in a dramatic depolarization of fluorescence. Only small changes in either the absorbance spectrum or fluorescence lifetime of the chromophore were detected upon FITC-C9 polymerization. Evidence is presented that the measured changes in FITC fluorescence upon C9 activation are due to self energy transfer between closely apposed fluorescein chromophores which occur in the polymerized form of the protein. The significance of these observations to the molecular structure of the assembled C5b-9 complex is discussed, as are the potential applications of this fluorescent derivative of C9.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003186:Complement C9 / Q000378:metabolism*; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism*; D006041:Goats / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008854:Microscopy, Electron; D011108:Polymers / Q000378:metabolism; D013861:Thiocyanates / Q000378:metabolism*
D003186:Complement C9; D015938:Complement Membrane Attack Complex; D005452:Fluoresceins; D011108:Polymers; D013861:Thiocyanates; D003165:Complement System Proteins; D016650:Fluorescein-5-isothiocyanate