还原型胰岛素A、B链相互作用的初步研究

本文报道在chance所用“一步法”重组胰岛素的类似条件下,还原型A、B链相互作用研究的初步结果。在pH10.6对S—磺酸型A及B链加入1.2倍SH/SSO_3—的二巯基苏糖醇,在一小时内A、B链与DTT的混合物较之单独的A、B链与DTT的混合物,紫外差光谱显示295、245nm负差峰,峰值随时间增大;园二色性测定观察到混合链的α螺旋含量比单独的A、B链有所增加。这表明在A、B链的巯基氧化生成二硫链前,还原型A、B链间的相互作用导致Tyr残基逐渐内埋,肽链构象的调整使有序二级结构增加。根据此时还原程度的测定,讨论了DTT与S—磺酸型A、B链作巯基交换的可能机制,过多的还原试剂易破坏已正确配对的二硫键,也妨碍肽链构象的调整。

Preliminary Studies of the Interaction of Reduced Insulin A and B Chains

In this work, preliminary results of the interaction of reduced insulin A and B chains have been reported under the conditions which were similar to those of chance "single step" method of reconstituting the active insulin from its reduced chains. After nearly stoichiometrie amounts of DTT to give a SH/SSO3 molar ratio of approximetely l.2 were added to the mixed S-sulfonated A and B chains as well as to the separated A or B chain at PH 10.6, the mixture of the chains show a difference spectrum against the separated chains with negative peaks at 245 and 295 nm which indicate a partial transfer of Tyr residues from an exposed to a buried state before the formation of the disulfied bridges from the reoxidation of the reduced chains. CD measurements indicate that the α-helix contens of mixed A and B chains in the presence of DTT increases in comparison with those of the separated chains with added DTT. It seems that upon mixing of reduced A and B chains,the chains fold gradually so as to interact through secondary forces leading to a confo-rmational read justment of polypeptide chains. It is the correct pairing of the chains which makes the successful resynthesis of insulin possible during the reoxidation of the reduced chains in chance method. According to the measurements of the reduction contents, possible mechanisms of disulfide exchange of S-sulfo'nated chains with DTT were discussed.