PRESSURE-INDUCED DISSOCIATION OF FLUORESCEIN FROM THE ANTIFLUORESCEIN SINGLE-CHAIN ANTIBODY 4-4-20

Hydrostatic pressure was used to dissociate fluorescein (Fl) from the high-affinity anti-Fl single-chain antibody 4-4-20 (SCA 4-4-20). Fl dissociation was monitored by measuring (1) the shift in the Fl absorption peak, (2) the recovery in Fl fluorescence intensity, which is quenched upon SCA binding, or (3) the decrease in Fl fluorescence polarization. Pressure effects were studied at two different Fl:SCA 4-4-20 molar ratios: 1:1, at which Fl fluorescence quenching was ca. 35% at atmospheric pressure, and 1:5, at which quenching reached 95-97% under the same conditions. In both cases, pressure-induced dissociation was favored by concomitant dilution of protein and ligand. Dissociation constants (K(D)) at each pressure were calculated on the basis of measurements of Fl fluorescence polarization under pressure. The dependence of K(D), and consequently of DELTAG of dissociation, on pressure permitted calculation of the magnitude of the standard volume change (DELTAV) involved in the dissociation process. According to this study, DELTAV of dissociation for the Fl-SCA complex is -50 mL/mol, which corresponds to a 10-times higher value than that found for dissociation of Fl from the intact IgG mAb 4-4-20 [Herron, J. N., Kranz, D. M., Jameson, D. M., & Voss, E. W., Jr. (1986) Biochemistry 25, 4602-4609]. This difference is explained in terms of a higher overall flexibility of unliganded SCA and of a less stable binding site in SCA relative to mAb. This interpretation was based on the findings that the Fl ligand stabilizes SCA conformation, that Fl dissociation was not determined by primary conformational changes in SCA structure induced by hydrostatic pressure, and that Fl is more accessible to the charged quencher iodide when liganded to SCA than when bound to mAb. Finally, the strong temperature dependence for pressure-induced dissociation of Fl from SCA 4-4-20 combined with the fact that association of the Fl hapten to SCA was found to be enthalpically unfavorable indicates that formation of the Fl-SCA 4-4-20 complex is an entropy-driven process.