LIGAND-BINDING TO ANTI-FLUORESCYL ANTIBODIES - STABILITY OF THE ANTIGEN-BINDING SITE

The problem of protein stability is addressed with spectroscopic studi es of equilibrium and kinetic properties of the binding of fluorescein to high-affinity monoclonal anti-fluorescyl antibodies (Mab 4-4-20), Fab fragments, and single-chain antibodies (SCA). SCA molecules contai n only the variable domains of the antibody and an amino acid linker. The influence of glycerol on the antigen binding reaction is studied b y circular dichroism, fluorescence, and absorption spectroscopy. The p resence of glycerol in the solvent lowers the affinity of SCA for the ligand drastically, and the affinity even decreases toward lower tempe ratures. This effect is not observed in Fab and Mab. Analysis of the t emperature jump kinetics shows that the dissociation reaction can be m odeled as a two-state transition. The CD spectra indicate that the dom ain structure of the SCA remains unaltered in the presence of glycerol . Therefore, it is concluded that glycerol promotes the dissociation o f the two variable domains of SCA. In Fab and Mab, the constant domain s provide additional stabilization of the molecular structure at the a ntigen binding site.