Chemical modification of alpha-bungarotoxin with bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene

alpha-Bungarotoxin (alpha-BuTX) was modified with a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene (DFDNB) to prepare the cross-linked derivatives. The modified products, obtained from the reaction at pH 8.5 with a 2-fold molar excess of DFDNB, were purified successively on a Sephadex G-50 and a SP-Sephadex C-25 columns, and, fast protein liquid chromatography (FPLC) on a Mono S column. The preparation was proved to be a homogeneous and dimeric form which arose from the intermolecular cross-linkage of alpha-BuTX through a dinitrophenylene (DPE) group. The cross-linked alpha-BuTX derivative still retained approximately 50% residual lethality and 70% residual binding activity to nicotinic acetylcholine receptor (nAChR). The modified derivative also showed relatively high antigenic activity toward anti-alpha-BuTX antibody as measured by competitive radioimmunoassay (RIA).