Temperature and pH dependence of fluorescein binding within the monoclonal antibody 9-40 active site as monitored by hydrostatic pressure.
In a comparative study, the thermodynamic parameter, DeltaV, was obtained using hydrostatic pressure-induced dissociation of fluorescein (Fl) from the active site of monoclonal antibody (mAb) 9-40 and its mutant and native derivatives equilibrated at six pH values (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5) and four temperatures (35, 25, 15, and 5 degrees C). mAb 9-40 and its Fab and single-chain Fv (scFv) derivatives at pH 8.0 were found to have identical Fl dissociation behavior under pressure as a function of temperature. The pressure dissociation at 25 degrees C as a function of pH showed a sigmoidal dependence of DeltaV with a midpoint value at pH 7.4 for mAb 9-40. Comparison of experimental results for scFv 9-40/212 with its mutant scFv 9-40/212Arg-34L indicated that the pH dependence of mAb 9-40 was due to the titration of His-34L in the active site. Iodide quenching of bound Fl showed that the hapten in this active site was solvent accessible. Imperfect packing, which leads to increased conformational dynamics, was determined as a possible cause of the low affinity for mAb 9-40.
Carrero
J
J
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, 61801-3797, USA.
Voss
E W
EW
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007454:Iodides; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D008962:Models, Theoretical; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007454:Iodides; D008024:Ligands; D019793:Fluorescein