title: Complement C4bC2 complex formation: an investigation by surface plasmon resonance.
abstract: Complex formation between the human complement proteins C4b and C2 was investigated by surface plasmon resonance. C4b was immobilised and C2 was used in the fluid phase to measure interaction at different ionic strengths (30-830 mM NaCl) and in the absence and presence of MgCl2. Maximum binding was observed at 30 mM NaCl, and was negligible above 300 mM NaCl. Binding was not greatly influenced by variation in Mg(2+) in the range of 2.5-15 mM. C4bC2 affinity (Kd) was determined by steady-state analysis to be 7.2x10(-8) M in physiological conditions (10 mM Hepes, 2.5 mM MgCl2, 0.75 mM CaCl2 and 140 mM NaCl, pH 7.4). For C4(H2O)C2 complex formation, a Kd of 4.0x10(-8) M was calculated. As far as detected by the applied method, complex formation does not involve conformational changes of one of the binding partners. Consistent with previous reports, C4bC2 binding takes place as a multiple-site binding event in the presence of Mg2+. C4bC2 complex formation in 10 mM Hepes, 2.5 mM EDTA and 140 mM NaCl (pH 7.4) was also observed and the interaction showed characteristics of a single-site binding event. Kd was 1.5x10(-8) M. Complement factor B (FB) was also tested for its binding to immobilised C4b. Weak interaction was observed at FB concentrations in the physiological range (500-1000 nM). Kd was 1.2x10(-6) M, indicating possible cross-reactivity between classical and alternative pathways of the activation of the complement system.
authors.affiliation: MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, UK. alaich@bioch.ax.ac.uk
authors.forename: A
authors.initials: A
authors.lastname: Laich
authors.forename: R B
authors.initials: RB
authors.lastname: Sim
mesh_terms: D003181:Complement C4 / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009994:Osmolar Concentration; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D003181:Complement C4; D020033:Protein Isoforms