SILICA-BASED METAL CHELATE AFFINITY SORBENTS .2. ADSORPTION AND ELUTION BEHAVIOR OF PROTEINS ON IMINODIACETIC ACID AFFINITY SORBENTS PREPARED VIA DIFFERENT IMMOBILIZATION TECHNIQUES

The chromatographic characteristics of some model proteins on silica-based metal chelates run under various experimental conditions are described. The retention of proteins on silica-based iminodiacetic acid (IDA) was compared among the different immobilization methods employed and to chelates bound on soft gels, such as chelating Sepharose and epibromohydrin-activated Sepharose. All Cu(II)-loaded chelators displayed adsorption of proteins in the presence of 0.5-1 M NaCl at pH 7-8; however, elution in the pH and imidazole gradient varied with the immobilization chemistry. The chemical structure in the neighbourhood of the metal chelate was of influence on the development of a negative charge with increasing pH. Thus, a negative charge evolves at lower pH with alkyl- than epoxy-immobilized IDA:Cu(I!). Glycidoxypropyltrimethoxysilane-immobilized IDA displayed almost identical chromatographic characteristics compared to chelating Sepharose FF. Basic proteins displayed higher retention on butyl-immobilized IDA compared to other chelates; interactions with positively charged amino acid residues seem to be superimposed on the interaction with histidyl residues. Proteins were least retained on 1,1'-carbonyldiimidazole-immobilized IDA:Cu(II); however, the selectivity for human and bovine serum albumins against other proteins employed was highest. The results obtained indicated that chelating interaction with some proteins depend on the spacer length. Thus, less flexible histidyl residues at protein surfaces might not be recognized from chelates immobilized by short spacers.