Circular dichroism of hapten-antibody complexes: Studies of tryptophanyl residue involvement in the antibody combining sites of MOPC-315 protein heavy and light chain subunits, reconstituted MOPC-315 protein and its Fv fragment

Abstract

Circular dichroic spectra of the native MOPC-315 mouse IgA myeloma protein and the protein reformed from its isolated heavy and light chains, the amino-terminal variable region fragment (Fv-315) and the isolated subunits provide information about conformational features of the isolated chains, changes which occur upon subunit association and about local protein-protein interactions. A pair of negative peaks in the near-ultraviolet (at 285 and 293 nm) is present in both the MOPC-315 and Fv-315 CD spectra, indicating that the structure responsible for the bands is located in the variable region of the intact immunoglobulin. These CD bands are absent from the CD spectra of the isolated heavy and light chain subunits but are regained in the process of subunit reassociation which results in the formation of the intact antibody combining site. The near-u.v. circular dichroism of a model cyclic tryptophanyl dipeptide provides evidence that two or more closely juxtaposed tryptophanyl residues may be responsible for the protein aromatic chromophore transitions at 285 and 293 nm. Binding of chromophoric haptens (Dnp- and Tnp-aminocaproate) to the native and reformed MOPC-315 protein and to its Fv fragment generates an induced circular dichroism with unique features: (1) an increase of the negative CD at both 285 and 293 nm; (2) a perturbation (blue-shift) of the tryptophanyl band at 293 nm; and (3) an apparent reciprocal relationship between positive induced CD bands of haptenic chromophore (at 370–382 nm) and the large negative induced CD band in the aromatic residue absorption region of the protein (near 300 nm) where haptens display absorption minima. These spectral features imply that the induced CD arises from electronic interactions between the aromatic moiety of the bound hapten and tryptophan(s) in the protein. This pattern of induced CD furnishes additional evidence that the paired CD bands at 285 and 293 nm in the spectra of the intact immunoglobulin and its Fv fragment arise from closely situated tryptophans and indicates that these residues lie in or near the intact antibody combining site. Induced CD bands generated by binding Tnp-aminocaproate to the isolated light chains also exhibit reciprocal relations and may be due to electronic interactions between the aromatic moiety of the bound hapten and tryptophan(s) in the protein subunit. Data available from primary amino acid sequence and affinity-label studies are consistent with the suggestion that variable region tryptophans from different subunits are close neighbora in or near the intact antibody combining site. The negative 293 and 285 nm CD bands of the MOPC-315 and Fv-315 proteins may therefore represent short range electronic interactions occurring within an antibody combining site between chromophoric residues contributed respectively by separate polypeptide chains.