Studies on the status of amino groups in alpha-bungarotoxin

The positive charges of amino groups in alpha-bungarotoxin (alpha-BuTX) were neutralized by reaction with trinitrobenzene sulfonate (TNBS) and were converted into negative charges with 4-chloro-3,5-dinitrobenzoate (CDNB). Three derivatives monotrinitrophenylated (TNP-) at Lys-38, 64, or 70; three di-TNP at Lys-38 and 64, Lys-38 and 70, and Lys-64 and 70; one tri-TNP at Lys-38, 64 and 70; and one penta-TNP at Lys-38, 51, 52, 64 and 70 as well as one mono-carboxydinitrophenylated (CDNP-) at Lys-38; di-CDNP at Lys-38 and 70, and tri-CDNP at Lys-38, 64 and 70 were isolated, respectively. The epsilon-amino groups at Lys-38, 64 and 70 are the most accessible to trinitrophenylation, Lys-51 and 52 are less reactive, while the N-terminus and Lys-26 are the least reactive. Each mono-TNP and CDNP derivative showed approximately 50% residual binding activity to nicotinic acetylcholine receptor (AChR)-rich membranes isolated from Torpedo californica and 50% of the lethality of alpha-BuTX. However, the activities were progressively lost as the accumulative modifications proceeded, and led finally to the formation of almost inactive penta-TNP derivative. The antigenicity of alpha-BuTX was still retained essentially intact after one or two amino groups of lysine residues were modified, whereas tri-TNP and CDNP-derivatives modified at Lys-38, 64 and 70 lost 46 and 70% of their antigenicity, respectively. Pronounced alteration in antigenicity was observed after five amino groups were trinitrophenylated. The present study indicates that the amino groups in alpha-BuTX may participate in the multipoint contact between the toxin and AChR, but none of the individual amino groups is definitely essential for the binding.