Spin-label studies of hapten combining sites in anti-p-azobenzoate antibodies.

Abstract The structural nature of the hapten combining site for anti- p -azobenzoate from four rabbits has been studied using spin-labeled benzoate haptens in which, (a) the spin-label (nitroxide free-radical) was attached directly to the p -aminobenzoate or with a spacer residue, viz., glycyl, β-alanyl or γ-aminobutyryl and (b), the spin-label was attached directly to m -aminobenzoate, o -aminobenzoate or to o -(methylamino) benzoate. Studies using the haptens under (a) showed that the rotational-freedom of the bound spin-label for the four preparations was increased with increased spacing and that although, for two of the preparations, the binding constant was decreased slightly by the introduction of the glycyl spacer, it increased with increasing spacer length. Of the haptens under (b), the o -(methylamino) benzoate derivative was not bound, presumably because the carboxylate group was displaced out of the plane of the benzene ring. On the other hand, the o -aminobenzoate derivative was measurably bound, presumably because the carboxylate group forms an intramolecular hydrogen bond to the amino group and is coplanar with the benzene ring. The meta -derivative was more strongly bound than the ortho -derivative but both the ortho - and meta-derivatives were bound less than the unspaced- p -aminobenzoate derivative. In the cases of both ortho -and meta-derivatives the spin-label was strongly immobilized indicating a tight fit for p -azobenzoate around these positions as was also indicated by the low binding of these compounds.