Changes in Intrinsic Circular Dichroism of Several Homogeneous Anti-Type III Pneumococcal Antibodies on Binding of a Small Hapten

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Abstract
Three homogeneous antibodies against the capsular polysaccharide of Type III pneumococci of similar specificities and affinities were purified from a single bleeding of an individual rabbit and fractionated by isoelectric focusing. A comparison of the circular dichroic spectra of the three antibodies revealed differences among them, although the spectra were generally similar to those obtained previously for heterogeneous rabbit antibodies [Cathou, R. E., Kulcycki, A., Jr. & Haber, E. (1968) Biochemistry 7, 3958]. On binding of the hexasaccharide, -[-->3)-beta-D-glucuronic acid-(1-->4)-beta-D-glucose-(1-](3)-->, significant changes in all three circular dichroic spectra were observed. Since the oligosaccharide alone shows no transitions above 220 nm, these spectral changes can be attributed to changes in the intrinsic optical activity of the antibodies. Calculated difference circular dichroism spectra (of antibody minus that of antibody-hapten complex) of the three antibodies are different from each other, and resemble spectra of tryptophan and tyrosine derivatives. These changes in optical activity can be ascribed to changes in the asymmetric environments of aromatic chromophores directly in the combining site and/or to changes in orientation inside or beyond the site. Since the hapten-antibody interactions are different in the three antibodies, as shown by the difference spectra, the structures of the combining sites are presumably also different. We have interpreted these observations to mean that a relatively simple ligand may be bound by several different complementary sites.