PRESSURE-INDUCED DISSOCIATION OF FLUORESCEIN FROM THE ANTIFLUORESCEINSINGLE-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 di ssociation was monitored by measuring (1) the shift in the Fl absorpti on peak, (2) the recovery in Fl fluorescence intensity, which is quenc hed upon SCA binding, or (3) the decrease in Fl fluorescence polarizat ion. Pressure effects were studied at two different Fl:SCA 4-4-20 mola r ratios: 1:1, at which Fl fluorescence quenching was ca. 35% at atmos pheric pressure, and 1:5, at which quenching reached 95-97% under the same conditions. In both cases, pressure-induced dissociation was favo red by concomitant dilution of protein and ligand. Dissociation consta nts (K(D)) at each pressure were calculated on the basis of measuremen ts 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) i nvolved in the dissociation process. According to this study, DELTAV o f 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 fro m 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 differ ence is explained in terms of a higher overall flexibility of unligand ed 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 pressur e, and that Fl is more accessible to the charged quencher iodide when liganded to SCA than when bound to mAb. Finally, the strong temperatur e dependence for pressure-induced dissociation of Fl from SCA 4-4-20 c ombined with the fact that association of the Fl hapten to SCA was fou nd to be enthalpically unfavorable indicates that formation of the Fl- SCA 4-4-20 complex is an entropy-driven process.