HIGH-PERFORMANCE METAL CHELATE INTERACTION CHROMATOGRAPHY OF PROTEINS WITH SILICA-BOUND ETHYLENEDIAMINE-N,N'-DIACETIC ACID

Wide-pore microparticulate silica gel having surface bound ethylenediamine-N,N′-diacetic acid (EDDA) was developed for the separation of proteins by metal chelate interaction chromatography (MIC). The separation of proteins including glycoproteins was carried out by gradient elution using increasing salt concentration. Different selectivities were obtained when changing the nature of the chelated metal on the surface of the stationary phase, manipulating eluent pH and varying the nature and concentration of the salts in the eluent. The retention-pH dependency of iron-free and holo transferrins suggested that MIC could be used for studying metal-protein complexes provided that the metal binding site in the protein molecule is also involved in the protein intractions with the chelated metal on the surface of the stationary phase. The eluting strength of sodium salts with "hard" metal-EDDA columns increased in the order Cl- ×CH3COO- ≤HCOO- ×H2PO-, whereas with "soft" metal-EDDA columns it increased in the order CH3COO- ×H2PO4- ×Cl-. Compared to another type of metal interaction column such as silica-bound iminodiacetic acid (IDA)k, the EDDA column exhibited different selectivity and retentivity toward proteins under other wise indentical elution conditions. Metal-EDDA stationary phases can be viewed as contemplementary to metal-IDA ones. © 1990.