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  • Title: Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody.
    Author: Blake RC, Li X, Yu H, Blake DA.
    Journal: Biochemistry; 2007 Feb 13; 46(6):1573-86. PubMed ID: 17279622.
    Abstract:
    Detailed equilibrium binding studies were conducted on a monoclonal antibody (8A11) directed against UO22+ complexed with 2,9-dicarboxy-1,10-phenanthroline (DCP-UO22+). Covalent modification of 8A11 with amine-reactive derivatives of Cy5 or Alexa 488 altered the binding curves obtained with DCP-UO22+ from hyperbolic to sigmoidal, the latter characterized by Hill coefficients of 1.5-1.6. Binding curves obtained with DCP-UO22+ and the bivalent (Fab)2 or the monovalent Fab fragments derived from limited proteolysis of the covalently modified 8A11 were characterized by Hill coefficients of 1.2 and 1.0, respectively. Incubation of 8A11 with saturating concentrations of the Fab fragments of goat antibodies directed against the Fc portion of mouse IgG increased the affinity of the native 8A11 for DCP-UO22+ by 3-fold. Conversely, incubation of the 8A11-Cy5 covalent conjugate with saturating concentrations of protein G (which likewise binds to the constant regions of mouse IgG) decreased the affinity of the primary antibody for DCP-UO22+ by 4-fold. In addition, the binding curves obtained with 8A11-Cy5 and DCP-UO22+ species changed from sigmoidal to hyperbolic at high concentrations of protein G. The presence of the antigen had a reciprocal effect on the binding of protein G to the 8A11-Cy5 conjugate; incubation of the 8A11-Cy5 conjugate with saturating concentrations of DCP-UO22+ decreased the affinity of the conjugate for protein G by 20-fold. These complex binding data were interpreted in terms of a free energy binding model in which (i) 2 mol of DCP-UO22+ and 1 mol of protein G bind to each mole of the 8A11-Cy5 conjugate, (ii) binding of the first equivalent of DCP-UO22+ to the antibody promotes the binding of the second equivalent of antigen in the absence of protein G, and (iii) DCP-UO22+ and protein G oppose each other's binding to the antibody. This is the first detailed description of the energetic balance of reciprocal binding events among the antigen binding sites and distant points on the constant portion of an immunoglobulin.
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