反相高效液相层析在胰岛素结构与功能研究中的应用

采用一种简单的“甲醇-水-0.1％三氟醋酸”作为洗脱体系的反相高效液相层析对研究胰岛素结构与功能关系,以及A链和B链相互作用时所用的一些胰岛素衍生物进行了快速、灵敏、准确的分析和鉴定。 胰岛素的S-磺酸型A链和B链只经一次离子交换层析纯化,经鉴定结果表明是均一的。S-硫甲基型A链和B链基本上也是均一的。通过对胰岛素还原产物的分析确定了能保证胰岛素折分完全并对其重组较为有利的还原条件。去B链羧端五肽(B26—30)胰岛素,以及去B链羧端八肽(B23—30)胰岛素能同时与天然胰岛素清楚地分离。一级结构差别极小的猪胰岛素和牛胰岛素也可得到分辨。胰岛素的羧端缩短的B链和A链重组的定量分析表明B链羧端肽段不但对胰岛素分子的生物活力的体现,而且在A链和B链的正确重组中都起着重要的作用。

Applications of Reverse Phase High Performance Liquid Chromatography in Structure-Function Studies of Insulin

A number of insulin derivatives have been analysed and characterized by reverse phase high performance liquid chromatography using a simple elution system composed of methanol, water and 0.1% trifluoroacetic acid. This method is much more rapid, sensitive and accurate as compared to conventional methods.Homogeneous S-sulfonated A and B chains of insulin and nearly homogeneous S-thiomethylchains satisfactory for studies on interaction between the chains in solution have been obtained.Pocine and bovine insulins can be resolved. Despentapepti de (B26-30)snd desoctapeptide(B23-30) insulin are readily resolved from native porcine insulin.Results from reconstitution studies on B chains shortened at C-terminal with A chain indicate an importent role played by the C-terminal part of the B chain in biological activity and correct reconstitution of insulin molecule as well.