Lin S R, Chang C C
Department of Chemistry, Kaohsiung Medical College, Taiwan, R.O.C.
Toxicon. 1991;29(8):937-50. doi: 10.1016/0041-0101(91)90077-5.
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.
通过与三硝基苯磺酸（TNBS）反应中和α-银环蛇毒素（α-BuTX）中氨基的正电荷，并用4-氯-3,5-二硝基苯甲酸酯（CDNB）将其转化为负电荷。分别分离出了在Lys-38、64或70处单三硝基苯基化（TNP-）的三种衍生物；在Lys-38和64、Lys-38和70以及Lys-64和70处双-TNP的三种衍生物；在Lys-38、64和70处的一种三-TNP衍生物；在Lys-38、51、52、64和70处的一种五-TNP衍生物以及在Lys-38处的一种单羧基二硝基苯基化（CDNP-）衍生物；在Lys-38和70处的双-CDNP衍生物以及在Lys-38、64和70处的三-CDNP衍生物。Lys-38、64和70处的ε-氨基最容易被三硝基苯基化，Lys-51和52的反应性较低，而N-末端和Lys-26的反应性最低。每种单-TNP和CDNP衍生物对从加州电鳐分离的富含烟碱型乙酰胆碱受体（AChR）的膜显示出约50%的残余结合活性以及α-BuTX 50%的致死性。然而，随着累积修饰的进行，活性逐渐丧失，最终导致形成几乎无活性的五-TNP衍生物。在赖氨酸残基的一两个氨基被修饰后，α-BuTX的抗原性基本上仍保持完整，而在Lys-38、64和70处修饰的三-TNP和CDNP衍生物分别丧失了46%和70%的抗原性。在五个氨基被三硝基苯基化后，观察到抗原性有明显改变。本研究表明，α-BuTX中的氨基可能参与毒素与AChR之间的多点接触，但没有一个单个氨基对于结合绝对是必需的。
