title: Aromatic residues mediate the pressure-induced association of digoxigenin and antibody 26-10.
abstract: We have previously found that the complex between fluorescently labeled digoxigenin and the monoclonal antibody 26-10 forms with a decrease in volume of approximately 30 ml/mol, leading to increased association of these species under applied hydrostatic pressure. In the present study, we have utilized a panel of mutant antibodies and Fab fragments, previously characterized for their importance in the binding affinity of digoxin:26-10, to probe the molecular basis of pressure sensitivity in this complex, as measured by fluorescence polarization spectroscopy. Several mutations that result in marked decreases in affinity exerted little or no significant effect on the association volume. Mutation at any of several key aromatic residues of the 26-10 Fab heavy chain led to a decrease in the pressure-induced association, and two mutants with Trp-->Arg mutations at heavy chain residue 100 exhibited pressure-induced dissociation. The effect of charged groups was found to depend on their proximity to contacting aromatic groups. The ability to understand and control the pressure sensitivity of antigen-antibody complexes has numerous potential applications in immunoseparations and immunosensors.
authors.forename: P
authors.initials: P
authors.lastname: Roy
authors.forename: C M
authors.initials: CM
authors.lastname: Roth
authors.forename: M N
authors.initials: MN
authors.lastname: Margolies
authors.forename: M L
authors.initials: ML
authors.lastname: Yarmush
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D004076:Digoxigenin / Q000737:chemistry*; D006874:Hydrostatic Pressure; D016297:Mutagenesis, Site-Directed
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D004076:Digoxigenin