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, Delta V, was obta ined using hydrostatic pressure induced dissociation of fluorescein (F l) from the active site of monoclonal antibody (mAb) 9-40 and its muta nt 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 pressu re as a function of temperature. The pressure dissociation at 25 degre es C as a function of pH showed a sigmoidal dependence of Delta V with a midpoint value at pH 7.4 for mAb 9-40. Comparison of experimental r esults for scFv 9-40/212 with its mutant scFv 9-40/212(Arg-34L) indica ted 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.