Comparison by1H NMR of the hapten environment in the combining site of the dinitrophenyl binding IgA protein 315 and its fragments

This paper compares the combining site of the Dnp binding mouse IgA myeloma protein in the Fv and Fab fragments and in the intact IgA. This was done by monitoring the environment of the aromatic hapten protons of DnpO⁻ , Dnp-l-aspartate and Dnp glycine on binding to protein 315, using high resolution 270 MHz¹H NMR. The aromatic Dnp proton resonances undergo large upfield chemical shifts which can be measured directly. These shifts have been interpreted previously as arising from interactions of the hapten with aromatic residues in the combining site. These shifts are an intrinsic reporter of the environment of the hapten in the combining site for each hapten. The magnitudes of the shifts are similar for both the Fv and Fab fragments. The absolute magnitude of the shifts for the IgA could not be measured directly, but the shift ratios for each Dnp proton were found to be similar to those obtained for the Fv and Fab fragments, indicating that the combining site is the same in all three fragments. The line-widths of the bound Dnp proton resonances vary with fragment size and show that the constant regions impose rotational constraints on the combining site. This is in contrast to the rotational freedom of the CH₃ domain which appears to be independent of the rest of the molecule (Burtonet al., 1977).