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Sequential Appearance of Three Different Anti-Fluorescein Combining Sites in Hyperimmunized Rabbits: Characterization by Circular Dichroism and Binding Studies

Department of Biochemistry and Pharmacology, Tufts Medical School, Boston, Massachusetts 02111

Abstract

Fluorescein (F1) bound in the combining site of anti-F1 antibodies exhibits extrinsic optical activity which is a function of the fine structure of the site. We have utilized this phenomenon to study structural changes in antibody combining sites as a function of time and repeated boosting. Four rabbits were immunized with F1-keyhole limpet hemocyanin (KLH) and successively boosted whenever the antibody production declined. Antibodies were purified from bleedings taken once or twice a week and characterized by the extrinsic circular dichroism (CD) of their hapten-antibody complexes. After the third boost, three characteristically different CD spectra were observed, each corresponding to a different combining site configuration.

Usually each individual bleed contained only one of the three antibody types and the particular type observed depended on the time after boosting: type I was present for the first 3 to 6 weeks of the antibody response, type II was present for a further 2 to 3 weeks and, finally, as the antibody production declined, only type III antibodies were found. This sequential response was repeated after each succeeding boost.

Each of the antibody types was functionally homogeneous with respect to its extrinsic CD properties. In contrast, binding data showed that they were somewhat heterogeneous. The induced CD bands of bound F1 originate in the xanthenone ring whereas the binding of F1 depends on the interactions of both the xanthenone and phenylcarboxylate moieties with the site. Therefore, these results suggested that the anti-F1 combining site consists of two distinct subsites. The functional homogeneity of each antibody type may be due to a common tertiary structure of the xanthenone-binding subsite whereas the heterogeneity indicated by the binding data may be due to heterogeneity within the phenylcarboxylate-binding subsite.

Possible mechanisms for the sequential appearance of these antibody types are also discussed.

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