Pressure-induced dissociation of fluorescein from the anti-fluorescein single-chain antibody 4-4-20

Abstract

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 (KD) at each pressure were calculated on the basis of measurements of Fl fluorescence polarization under pressure. The dependence of KD, and consequently of delta G of dissociation, on pressure permitted calculation of the magnitude of the standard volume change (delta V) involved in the dissociation process. According to this study, delta V 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of anti-fluorescent probe monoclonal antibody by long-chain amphiphiles

“…The quenching decreased with pressure as seen in the figure. In the previous paper [24], we have calculated the standard volume change associated with the dissociation of FL and IgG-49 from the FL-IgG-49 complex and obtained the value of −5.2 cm 3 mol −1 , that is, the complex expands slightly by binding of them. Similar dissociation behavior of anti-fluorescyl [25][26][27] and anti-BSA [28] monoclonal antibodies by pressure has been observed, and our value is close to the value reported for the anti-fluorescyl monoclonal antibody with a strong affinity to FL by Herron et al [15]. The value decreased with increasing temperature.…”

Comparative Properties of the Single Chain Antibody and Fv Derivatives of mAb 4-4-20

“…This indicated that Fv 4-4-20 displayed the same standard volume change (⌬ diss : Ϫ50 ml/mol) upon fluorescein dissociation as SCA (38). Seeing that their structures were apparently identical, this suggested that the Fv 4-4-20 must have increased conformational dynamics relative to the IgG molecule (⌬ diss : Ϫ5 ml/mol) as originally postulated for SCA (15,38,41). This indicated that increased dynamics were responsible for the decreased affinity for antigen displayed by Fv and SCA.…”

“…As previously stated, hydrostatic pressure does not promote changes on the tertiary structure of proteins, but alters regions of secondary structure responsible for global protein conformation (38,66). A comparison of the pressure induced dissociation of fluorescein profiles for Fv and SCA would be a definitive evaluation of their dynamic similarity.…”

“…mAb 4-4-20 was a suitable antibody for this study due to its high degree of structural characterization (7,33,34) and the previous construction and characterization of SCA 4-4-20 (7,35). SCA 4-4-20 has been studied extensively in terms of active site environment (36,37), antigen binding structure (13), and thermodynamics (8,38). Comparison of SCA with Fab 4-4-20 showed almost identical guanidine-induced denaturation profiles, idiotype and metatype expression, yet a 2-3-fold reduction in affinity for fluorescein (7).…”

Biocompatibility and Structural Stability of a Laminin Biopolymer

“…Molecular interactions destabilized by cold are those driven by the entropic component of the free energy of association, [22,23] whereas, this entropic component is usually derived from the favorable contribution of the hydrophobic effect. [24] Laminin self-polymerization in neutral pH is an entropically driven process.…”