Roles of the aromatic residues in cobrotoxin in antigenicity and binding activity to nicotinic acetylcholine receptor.

The single Trp-29 in cobrotoxin was modified with 2-hydroxy-5-nitrobenzyl bromide, and Tyr-25 and -35, with tetranitromethane. Although the binding activity of cobrotoxin to the nicotinic acetylcholine receptor (nAChR) was decreased after modification of Trp-29, the antigenicity remained essentially unchanged as measured by a competitive RIA. Tyr-35-nitrated cobrotoxin still retained relatively high antigenicity and potent binding activity to nAChR. However, modification of both Tyr-25 and Tyr-35 led to an altered secondary structure, with greatly reduced binding to antibody and receptor. Hence, Tyr-25 in cobrotoxin is essential for the maintenance of the active conformation for the bindings. Although the non-precipitating antibody against cobrotoxin exhibiting a higher neutralizing capacity than did the precipitating antibody, the binding affinity of the former to cobrotoxin and its modified derivatives was lower than that of the latter.