N.m.r. investigation of hapten binding to the myeloma protein M460

The binding of the haptens DnpOH, Dnp-lysine and Dnp-aspartate to the mouse myeloma IgA protein was studied using ¹H 270 MHz nuclear magnetic resonance spectroscopy. The n.m.r. difference spectra showed fewer resonance perturbed than expected. This is explained in terms of chemical exchange between the T and R states of the protein as described by the kinetic scheme of Lancet and Pecht (Lancet, D. and Pecht, I. Proc. Natl Acad. Sci. USA 1976, 73 3549 53). Large upfield chemical shifts were observed on the resonances of the hapten DnpOH on binding to M460. These are interpreted as indicating an aromatic environment for the Dnp ring. In contrast, the Dnp-aspartate resonances were not shifted at all, as would be expected from the observed rate constants using the kinetic scheme. The shifts observed on the hapten Dnp-lysine were much smaller than those observed for DnpOH. A range of possible values of the shifts were calculated for the T and R states, for Dnp-lysine and DnpOH. For both haptens the combining site environment differed between the T and R conformational states of M460, suggesting that the conformational change involves the combining site.