Antibody-hapten interactions in solution

Abstract

This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150000, but enzymic digestion yields the Fv fragment of molecular mass 25000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pK _a values of about 8.1, 6.9 and 6.1. The histidine resonances with pK _a values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102 _H and His 97 _L . The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd hi water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd hi. At pH 5.5 there is one tight binding site for the lanthanides (K _D ≈ 80 μm) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd hi quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.