Nakagawa Y, Yip T T, Belew M, Porath J
Institute of Biochemistry, University of Uppsala, Sweden.
Anal Biochem. 1988 Jan;168(1):75-81. doi: 10.1016/0003-2697(88)90012-7.
The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.
本文描述了在高效固定化金属离子亲和色谱柱上对30多种生物活性合成肽及其类似物的分离。金属螯合凝胶（TSK凝胶螯合物-5PW）含有与珠径为10微米的亲水性树脂基基质共价偶联的亚氨基二乙酸（IDA）。肽在固定于螯合凝胶上的铜（II）、镍（II）和锌（II）离子上的保留情况表明，其中一些肽可以通过等度洗脱分离，而大多数肽被保留，并通过线性咪唑梯度或连续降低的pH梯度洗脱分离成不同的馏分。在这里研究的三种固定化金属离子中，无论使用何种梯度类型，IDA-铜（II）螯合柱都具有最佳的分离度。血管紧张素I和II及其七种合成类似物的分离充分说明了这一点。本文获得的结果为今后利用这种分离方法在分析或制备规模上对多种肽进行有效分级分离提供了指导。
