Salt-induced immobilization of small affinity ligands on an epoxide-activated affinity support

The salt-induced immobilization of small ligands onto an epoxy-activated HPLC stationary phase was found to be an effective route to the preparation of HPLC-compatible affinity supports. The ligands S-methylglutathione, S-butylglutathione and S-octylglutathione were immobilized at pH 10.5 under various conditions of salt type and concentration. In potassium carbonate buffer the extent of reaction for the alkylglutathione ligands was insensitive to carbonate concentration (0.5-2 M) but was substantially influenced by the hydrophobicity of the ligand. S-Octylglutathione was immobilized with a 4-fold greater efficiency in 2 M potassium carbonate than was the less hydrophobic S-methylglutathione. By contrast, in potassium phosphate buffer the extent of the immobilization was strongly dependent on salt concentration. For the S-alkylglutathiones, a 2-fold increase in immobilization efficiency was found when increasing the phosphate concentration from 0.5 to 2 M. Prior theory was extended to explain these results involving a salt-induced hydrophobic interaction between the ligand and the epoxy phase which in turn results in faster reaction rates between ligand and epoxy groups. As predicted by this model, the immobilization of the very hydrophilic ligand, nitrilotriacetic acid, was found to be affected minimally by either salt type or salt concentration. Further, when the rate of hydrolysis of the immobilized epoxy groups was studied, the maximum amount of immobilized ligand was found, in some cases, to be limited by the hydrolysis of the epoxy groups. Practical application of these principles allowed a nickel chelate HPLC-affinity phase to be produced using the nitrilotriacetic acid as the ligand. The HPLC phase was found to be useful in multiple, small-scale purifications of a histidine-tagged protein kinase C fragment. (C) 1998 Elsevier Science B.V.