Binding dynamics in biological systems. Interaction of MOPC‐315 with ¹⁹F labelled nitrophenyl haptens

Abstract

We have studied the dynamics of binding of ' % 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 afhity (K-1O3M-') site@) 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 I & ' ) , agree with on-rates determined at much lower protein concentrations ( 5 1 mg nd-'); we therefore conclude that protein aggregation, if it does occur, does not significantly affect binding in these systems.Recognition? and binding of antigen by antibody occurs as a first step in the response of the immune system to a foreign invader. Interaction between antibody and antigen (or hapten) has been studied by a variety of methods; most recently NMR techniques have proved useful in obtaining information about binding affinities and the environments experienced by bound ligands in the case of several antibodies specific for haptens related to phosphorylcholinel.* and of MOPC-315 (both the intact 7s immunoglobulin and Fab' and Fv fragments) which specifically binds nitrophenyl hap ten^.^-' With MOPC-315, these studies demonstrated that the binding pocket of the 7S, Fab' and Fv proteins provides the same environment to bound * Author to whom correspondence should be addressed.t Abbreviations used are: k,,, off rate; k,,, on rate; A, total chemical shift (separation of NMR absorption of the bound and free hapten) measured in Hz; 6, observed chemical shift (the actual position of the NMR signal which depends on the mole fraction of the ligand bound); B, subscript used to represent hapten bound; F, subscript used to represent free unbound hapten; P, mole fraction; 7, average life time which is equal to the weighted average of the life hapten and also that different nitrophenyl haptens with very different thermodynamic binding affinities are bound in the same orientation in the same site. The environment experienced by a trifluoromethyl group attached to a phenyl ring depends only on the structural relationship between the CF, group and the side chain of the hapten and not on the nature of other phenyl substituents or on the structure of the side chain5In this paper, we explore the relative dynamics of binding o f nitrophen...