金属螯合亲和色谱中的疏水作用

通过考察盐溶盐和盐析盐浓度对蛋白质在IDA裸柱和金属螯合柱上保留行为的影响，详细研究了金属螯合色谱中的疏水作用，疏水作用的发生、形成的条件以及不同条件下对蛋白质保留值的贡献。实验结果表明，在高浓度和低浓度的盐溶盐以及低浓度盐析盐中，蛋白质在金属螯合柱上的保留主要受静电和配位作用控制，而疏水作用对蛋白质的保留影响很小。对弱亲和性的金属螯合柱以静电作用为主，其大小可用参数Q表征；对强亲和性的IDA-Cu（Ⅱ）柱以配位作用为主。仅在高浓度的盐析盐中，金属螯合柱才呈现较强的疏水作用，支配蛋白质保留。实验证明，金属螯合色谱中疏水作用主要来自固定相间隔臂中的疏水碳链和盐析盐对蛋白质的增疏作用，利用这种疏水作用有可能改善金属螯合色谱分离的选择性。

Hydrophobic Interaction in Metal Chelate Affinity Chromatography

The formation,producing condition and contribution to protein retention under different conditions of hydrophobic interaction have been studied in detail by(examining) the effects of salting-in salt and salting-out salt concentration on the retention behaviors of(protein) on naked iminodiacetic acid(IDA) and metal chelate column.The experiment showed that the retention of(protein) on metal(chelate) column was mainly controlled by electrostatic(interaction) and coordination role in high and low concentration salting-in salt,as well as low concentration salting-out salt,while the effects of hydrophobic interaction on the(protein)(retention) were quite small.The electrostatic interaction was the first for the metal(chelate) column with weak affinity.Its magnitude can be expressed as a parameter Q.The coordination role was main for the metal(chelate) column with strong affinity.Only in high concentration salting-out salt solution,the metal chelate column appeared a stronger(hydrophobic) interaction which can dominate the protein(retention).According to a control experiment for the hydrophobicity of chromatographic column,it was demonstrated that the hydrophobic interaction of metal(chelate) column resulted mainly from hydrophobic carbon chain in the spacer arm on stationary phase and(increasing) hydrophobic interaction of salting-out salt to the proteins.Using the hydrophobic interaction of metal chelate column in high concentration salting-out salt solution,the selection of chromatographic separation may be improved.