EFFECTS OF SECONDARY FORCES ON THE LIGAND-BINDING AND CONFORMATIONAL STATE OF ANTIFLUORESCEIN MONOCLONAL-ANTIBODY-9-40

Biochemical interactions that occur external to the antibody active si te have been termed secondary forces, Secondary forces are supplementa l to interactions within the antibody active site (i.e., primary inter actions) and can affect ligand binding efficiency as well as variable domain conformation. The antifluorescein antibody system has been dete rmined to be a superior method for delineating primary from secondary interactive components due to the active site-filling properties of th e fluorescyl ligand. To date, all studies of secondary forces within t he context of the antifluorescein system have been with the high-affin ity monoclonal antibody 4-4-20 (mAb 4-4-20) (Mummert & Voss, 1995, 199 6, 1997), In order to determine the generality of experimental observa tions and proposed models, we investigated the effects of secondary fo rces on the antifluorescein mAb 9-40. In addition to assessing the res ults of former studies, mAb 9-40 possesses properties unique from thos e of mAb 4-4-20, namely, a decreased affinity for fluorescein and incr eased conformational dynamics relative to mAb 4-4-20 (Carrero & Voss, 1996), Results of fluorescein and intrinsic mAb 9-40 tryptophan quench ing as well as differential scanning calorimetric (DSC) studies indica ted that secondary forces modulated the conformational (metatypic) sta te in accordance with previous investigations with mAb 4-4-20, Unlike mAb 4-4-20, mAb 9-40 did not exhibit altered ligand binding efficiency due to the inclusion of secondary interactive compoents. Model were d eveloped that proposed chat the increased malleability of mAb 9-40 var iable domains could account for functional differences in properties b etween mAb 9-40 and mAb 4-4-20.