金属螯合亲和色谱中固定金属与蛋白质的作用

Interaction between Immobilized Metal and Protein in Metal Chelate Affinity Chromatography

在不同pHNaCl的磷酸缓冲体系 ,比较了牛血清蛋白 (BSA)、核糖核酸酶 (RNase)、细胞色素C(Cyt C)和溶菌酶 (Lys)在IDA裸柱和一些金属螯合柱上的保留特性 ,考察了固定金属对蛋白质保留行为的影响。指出蛋白质在强结合IDA Cu柱上的保留主要受固定金属和蛋白质间配位作用支配。在弱亲和的IDA Ni、IDA Co和IDA Zn柱上的保留主要受静电作用控制 ,配位作用为辅。讨论了金属螯合亲和色谱中影响蛋白质和金属配位的主要因素 :金属离子的电荷和半径、配位原子对中心离子外层d轨道的影响 ,以及蛋白质表面配位的组氨酸数目、离解常数和取向。影响金属螯合配体和蛋白质静电作用的主要因素为溶液的pH和蛋白质的等电点pI。

Retention characteristics of bovine serum albumin(BSA), ribonuclease(RNase), cytochrome C(Cyt-c) and lysozyme(Lys) on iminodiacetic acid (IDA) bare columns and several metal chelate columns were compared at phosphate buffer of sodium chloride with different pH. The influences of the immobilized metal on retention behaviors of proteins were examined. It was proposed that the retention of proteins on IDA-Cu column with strong affinity was mainly dominatled by the coordination role between the immobilized metal and protein. The protein retention on IDA-Ni, IDA-Zn and IDA-Co column with weak affinity was mainly controlled by the electrostatic interaction between metal chelate ligand and protein, whereas the coordination role was additional in the protein retention. The factors influencing the coordination and electrostatic interaction between the protein and metal were discussed at different chromatographic conditions. The main factors influencing the protein coordination involve the charge and radius of metal ion, the influence of ligand on outer shell d-orbit of the center atom as well as the number, dissociation constant and orientation of the coordinative histidine at protein surface. The factors effecting the electrostatic interaction between the metal chelate ligand and protein are pH of the solution and pI of protein.