Pressure-induced dissociation of fluorescein from the anti-fluorescein 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 (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)
Coelho-Sampaio
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Department of Microbiology, University of Illinois, Urbana 61801.
Voss
E W
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D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D006874:Hydrostatic Pressure; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D000906:Antibodies; D005452:Fluoresceins; D019793:Fluorescein