title: Random and site-specific immobilization of catalytic antibodies.
abstract: The effects of immobilization on the immunologic and catalytic activity of a catalytic antibody were compared for randomly immobilized (via glutaraldehyde) whole antibody and site-specifically immobilized (via the reactive sulfhydryl group at the base of the fragment) Fab' fragments. Upon immobilization, the specific binding capacity (n) and the catalytic activity decreased significantly for both systems. Increases in the Michaelis constant (KM) were accompanied by corresponding decreases in the equilibrium binding constant determined through immunoassays. For the immobilized Fab', n decreased dramatically with increased protein loading, suggesting that, despite the site-specific attachment and favorable orientation, molecular crowding denatured the Fab' fragments. These results also show that there is an optimal surface coverage, not necessarily at the maximum loading, for both immunologic and catalytic activity. Finally, the combining/active site conformation was probed using electron paramagnetic resonance (EPR) spectroscopy. In all antibody samples, there was no spectral evidence of conformational changes in the antibody active site.
authors.affiliation: Department of Chemical Engineering, University of California, Berkeley 94720.
authors.forename: T M
authors.initials: TM
authors.lastname: Spitznagel
authors.forename: J W
authors.initials: JW
authors.lastname: Jacobs
authors.forename: D S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D000818:Animals; D017151:Antibodies, Catalytic / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D004800:Enzymes, Immobilized; D005976:Glutaral; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D012995:Solubility
chemical_list: D017151:Antibodies, Catalytic; D004800:Enzymes, Immobilized; D007140:Immunoglobulin Fab Fragments; D005976:Glutaral