Structural Studies on the Combining Site of the Myeloma Protein MOPC 315

Abstract

1 Analysis of the electron spin resonance spectra of different spin-labelled haptens when bound to the Fv fragment from the immunoglobulin A of the mouse myeloma protein MOPC 315 suggests that the combining site is a cleft of overall dimension 11 Å× 9 Å× 6 Å which has considerable structural rigidity. 2 The 270 MHz proton nuclear magnetic resonance spectrum of the amino acid residues in and around the combining site is obtained by use of paramagnetic difference spectroscopy involving a spin-labelled hapten. There are only the equivalent of about 30 aliphatic and 30 aromatic protons in this difference spectrum. 3 The C-2 and C-4 proton resonances of three histidine residues in the Fv fragment are observed to titrate with pH. The pKa values of these histidine residues are about 8.1, 6.9 and 6.1. The resonances of the histidine residue with pKa value 8.1 show anomalous behaviour in splitting into two or more components. The values of the chemical shifts of the C-2 and C-4 protons alter slightly in the presence of hapten, particularly for the histidine residues with pKa values of 6.9 and 6.1. The resonances of these two residues are not observable in the presence of the spin-labelled hapten indicating that these two histidines are in the region of the combining site. 4 The existence of lanthanide binding sites of the Fv fragment, an essential prerequisite in mapping studies, has been demonstrated by measurements of the solvent water relaxation rates in Gd3+ solutions.