Magnetic resonance studies of the binding site interactions between ^(19)F-labeled nitrophenyl haptens and specific mouse myeloma immunoglobulin MOPC-315

The interactions between MOPC-315, a mouse myeloma protein with specificity for nitrophenyl haptens, and ^(19)F-substituted haptens have been investigated using nuclear magnetic resonance (NMR) spectroscopy. The haptens studied are mono- or dinitrophenyl derivatives of γ-aminobutyric acid, lysine, or glycine which have trifluoromethyl groups attached to the phenyl rings. Upon binding to immunoglobulin, the ^(l9)F nucleus experiences a downfield shift whose magnitude depends on the position of the trifluoromethyl group on the phenyl ring but is independent of other structural changes in the hapten such as the number of nitro groups attached to the phenyl ring. Further, the chemical shift of bound hapten is not influenced by the amount of the constant region attached to the binding site; we accordingly conclude that the presence of the distal, constant regions of the immunoglobulin molecule does not influence binding site interactions

Binding Dynamics in Biological Systems. Interaction of MOPC-315 with ^(19)F Labelled Nitrophenyl Haptens

We have studied the dynamics of binding of ^(19)F labelled haptens by mouse plasmacytoma antibody MOPC-315, a protein which shows specificity for nitrophenyl haptens. The off-rates for the dissociation of hapten from MOPC-315 proteins (7S, Fab′ and Fv) were determined by the application of several methods including a technique which, in certain cases, allows the direct determination of the rate of exchange of nuclei between magnetically non-equivalent sites without requiring prior knowledge of intrinsic line widths. Used in conjunction with independently determined data on line widths, chemical shifts, and binding affinities, these studies show that the rates for hapten association to, or dissociation from, the intact 7S antibody, the Fab′ fragment, and the Fv fragment of MOPC-315 are essentially the same. They also indicate the presence, probably in the Fv region, of a low affinity (K∼ 10^3M^(−1)) site(s) for hapten binding. The mobility of the hapten combining site decreases as the size of the protein increases. These rate data, which were determined at relatively high protein concentrations (up to 40 mg ml^(−1)), agree with on-rates determined at much lower protein concentrations (≤1 mg ml^(−1)); we therefore conclude that protein aggregation, if it does occur, does not significantly affect binding in these systems