The distance between the contact sites for DNP and menadione ligands in the combining region of myeloma proteins binding both haptens—II: Estimation of distance using haptenic probes with variable length spacers

Abstract

Proteins 315 and 460 are IgA 2 mouse myeloma immunoglobulins which bind the haptens 2,4-Dnp and Menadione, and their derivatives competitively in the combining region. We have studied the binding of these proteins to a series of Dnp and Menadione based solid phase immunoabsorbents with different length spacer groups to which the Dnp and Menadione groups are attached, in order to determine how deep in the combining region each hapten binds. Both protein 315 and protein 460 share similar combining region dimensions with respect to the haptens used. Menadione binds in the combining regions of both proteins at a depth of 22 Å, while Dnp binds at a depth of 8.8 Å in protein 460 and 11 Å in protein 315. The difference between these values, 11–13 Å, measures the separations between the contact sites for these two haptens. In the preceding paper (pp. 929–937) calculations from energy transfer data gave an average minimum distance of 11.4 Å between the contact sites of Dnp and Men in the protein 460 combining regions. Thus, there is agreement between these distances obtained by two independent experimental methods.