title: Isolation of the C9b fragment of human complement component C9 using urea in the absence of detergents.
abstract: The bactericidal activity of the C5b-9 complex of complement is dependent upon the terminal complement component C9. The precursor C5b-8 complex is not harmful to bacterial cells until C9 is added to complete the C5b-9 complex. The C9 molecule can be proteolytically cleaved by thrombin to yield an intact, nicked molecule that remains fully functional when added to either bacterial cells or erythrocytes bearing pre-formed C5b-8 complexes. In investigating the membranolytic function of C9 in the C5b-9 complex, the carboxyl-terminal portion of the nicked molecule (C9b) has been shown to be membranolytic when added to erythrocytes, liposomes, or bacterial inner membranes in the absence of any other complement components. The isolation of C9b from nicked C9 has been accomplished by preparative gel electrophoresis using detergents, however the study of the activity of C9b in membrane systems may be complicated by the possible presence of residual detergent. To address this concern, we have used 4 M urea in conjunction with hydroxyapatite chromatography and a phosphate elution procedure to separate the domains of nicked C9. The isolated C9b domain, free of detergents and in the absence of any other complement components, was found to be membranolytic. C9b isolated in this manner was capable of lysing erythrocytes and inhibiting the growth of bacterial spheroplasts.
authors.affiliation: Department of Biology, University of Southwestern Louisiana, Lafayette 70504, USA.
authors.forename: X
authors.initials: X
authors.lastname: Gu
authors.forename: J R
authors.initials: JR
authors.lastname: Dankert
mesh_terms: D003186:Complement C9 / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification* / Q000502:physiology; D003902:Detergents; D006461:Hemolysis; D006801:Humans; D013104:Spheroplasts / Q000187:drug effects / Q000276:immunology; D014508:Urea
chemical_list: D003186:Complement C9; D003902:Detergents; C048666:complement C9b; D014508:Urea