title: Regional and segmental flexibility of antibodies in interaction with antigens of different size.
abstract: The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range.
authors.affiliation: Graduate School of Agriculture, Kyoto Prefectural University, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oda
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Kiichi
authors.initials: K
authors.lastname: Fukui
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Takachika
authors.initials: T
authors.lastname: Azuma
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism; D000941:Antigens / Q000737:chemistry* / Q000378:metabolism; D019540:Area Under Curve; D002417:Cattle; D002645:Chickens; D013058:Mass Spectrometry; D008970:Molecular Weight; D046912:Multiprotein Complexes; D010047:Ovalbumin / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
chemical_list: D000906:Antibodies; D000941:Antigens; D046912:Multiprotein Complexes; D012709:Serum Albumin; D010047:Ovalbumin