Combination of Specific Antibodies with the Human Vitamin A-transporting Protein Complex

The interactions of specific antibodies with the human retinol-binding protein (RBP) and the thyroxine-binding prealbumin have been investigated. When an antibody combines with either of these proteins, the tryptophyl fluorescence of the antibody is partially quenched. Polarization of fluorescence of RBP-retinol increased rapidly on formation of antibody-RBP complexes. With fluorescence-quenching and polarization measurements as indicators, the stoichiometries and equilibrium constants of the antigen-antibody reactions have been studied. The data show that RBP, whether free or in complex with prealbumin, exhibits identical reactivity with anti-RBP Fab′-fragments. The conclusion is reached that the prealbumin binding site of RBP is not a major antigenic structure. The data indicate that the number of antigenic sites on RBP is limited and is the factor which controls the number of antibodies bound per RBP molecule. By quantum yield measurements of retinol it was found that RBP does not undergo any measureable conformational change on complex formation with Fab′-fragments. Free prealbumin could simultaneously interact with a maximum number of 12 anti-prealbumin Fab′-fragments. This suggests that there are three antigenic sites on each of the four identical prealbumin subunits. RBP, on forming a complex with prealbumin, competed with four anti-prealbumin Fab′-fragments. These Fab′-fragments showed a limited heterogeneity indicating that they may be directed toward a single antigenic site. It is proposed that the competition between RBP and the anti-prealbumin Fab′-fragments may be interpreted as a consequence of a negative cooperativity on RBP forming a complex with prealbumin. The equilibrium constant for Fab′-fragments forming a complex with prealbumin was lowered on thyroxine binding to prealbumin. This result supports the earlier suggestion that thyroxine binding to prealbumin is an example of a negative homotropic interaction.