Immunochemical analysis of the neurotoxin apamin and its derivatives

The objective of the present investigation was to conduct an immunochemical analysis (radioimmunochemical analysis) of apamin using derivatives of the latter with modifications of various amino acid residues and antibodies against apamin. Immune serum was used for the analysis based on competition between the unlabeled and labeled ligand for the active sites of the antibodies. Since apamin does not contain tyrosine residues, for its iodination Lys4 derivative of apamin was first prepared, which was then radioactively iodinated with 125I using chloramine T. The 125I was removed from the labeled [125I]apamin by gel filtration on Sephadex G-10 and reprecipitation with saturated picric acid in the presence of an 0.2% histone solution. The following derivatives of apamin were used: 1) a Lys4 derivative disuccinylated on the Cys1 residues; 2) a Lys4 derivative diacetylated on the Cys1 residues; 3) the diacetylated derivative (Cys1, Lys4) containing carboxymethylated His18, obtained by treating His18 with diethylpyrocarbonate; 4) a derivative of apamin containing dipyrimidyl-Orn13, Orn14. Results show that unlabeled apamin has the greatest competitive activity with respect to [125I]apamin in the system [125I]apamin - antibodies against apamin and then in decreasing order of competitive activities (apamin deriviatives with the corresponding modifications): apamin > disuccinylated-Cys1, Lys4-apamin > diacetylated-Cys1, Lys4-apamin > diacetylated-Cys1, Lys4-carboxymethylated-His18-apamin. The derivative in which the Arg13 and Arg14 residues had been converted to dipyrimidyl-Orn13, Orn14 had the least activity. Since these two arginine residues of apamin are the residues that are most important for the neurotoxic effect of the toxin, it can be concluded that the antigenic determinant of apamin found in the system that we used is located close to or coincides with Arg13, Arg14