Histidine-ligand chromatography of proteins Multiple modes of binding mechanism

The objective of this work is to exploit the versatile applications of matrix-linked histidine in protein chromatography. The negatively charged histidine at alkaline pH acts as a cation exchanger for proteins with high isoelectric points. Because of the hydrophobicity of the imidazole ring in histidine, enzymes, such as yeast alcohol dehydrogenase, are strongly retained by this histidine-ligand column in presence of 1.2 M ammonium sulfate. Due to the high formation constants of complexes between immobilised-histidine and metal ions, this column could be applied to serve as a ligand-exchange chromatography. In this instance, the interaction between proteins and the ligand takes place via the coordination sphere of the complex-forming metal ions. All these binding modes have been elucidated by the investigations of the effects of pH, metal ions, salt type and concentrations on (1) the purification of trypsin and (2) the binding of model proteins. It should be noted that this work differs from the reported applications [M.A. Vijalakshmi, Trends Biotechnol. 7 (1989) 71] where the protein binding occurs at low ionic strength and at pH values near or around their isoelectric points