Reactivity of the antibodies to DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene

Abstract

Rabbits were immunized with native DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. The interactions between the purified antibodies to nDNA-AAF (or the Fab fragments) and several ligands have been studied. By radioimmunoassay, nDNA-AAF, dDNA-AAF, and GMP-AAF were found to bind to the antibodies with about the same affinity. GMP-AF interacts slightly less, and GMP and N-OH-AAF do not interact. The values of the association constants deduced from fluorescence measurements for the binding of the Fab fragments to nDNA-AAF, dDNA-AAF, and GMP-AAF, in 50 mM NaCl, pH 7.5, are of the same order of magnitude. The values of the association constants with nDNA-AAF and dDNA-AAF depend upon salt concentration. From this variation; it is deduced that 1-1.5 phosphate groups interact by charge--charge interactions with the Fab fragments. The absorption and circular dischroism spectra of GMP-AAF, nDNA-AAF, and dDNA-AAF bound to the Fab fragments show that the Fab fragments induce similar perturbation to the three ligands. These results lead to the conclusion that the immunodeterminant group is the dGMP-AAF residue.