Location of membrane-bound hapten with different length spacers.

Immunogenic activity of a lipid hapten is strongly dependent of the length and nature of the linker chain (spacer) connecting the hapten to the head group of the lipid. A derivative containing a very short or a long spacer is known to be less effective for antibody binding than that of an intermediate length. In the present experiment, this was confirmed first by experiments of antibody binding to TNP lipid haptens with different length of spacers and of antibody-dependent macrophage binding to them. Second, we determined the location of the TNP haptens in lipid bilayer membranes by fluorescence energy transfer. It was found that vertical distances between TNP groups (acceptors), which were assumed to be randomly distributed in a plan parallel to the membrane surface, and a pyrene fluorophore (donor), which was embedded in the middle of lipid membranes, were 10.2-10.5 A in the DMPC membranes and 13.2-13.9 A in the DPPC membranes. The vertical distances were about 3 A longer in the DPPC membranes than in the DMPC membranes. However, they were almost independent of the length of spacers. This indicates that TNP residues of the lipid haptens locate at the similar vertical position on the membrane surfaces even if they have different length spacers. From these results we suggested that the affinity of the spacer groups to the bilayer surfaces can modulate the binding affinity of antibody to lipid hapten on the membrane surfaces. This was partly supported by the binding experiments of TNP spacers to the bilayer membranes.