PROTEIN SELECTIVITY IN IMMOBILIZED METAL AFFINITY-CHROMATOGRAPHY BASED ON THE SURFACE ACCESSIBILITY OF ASPARTIC AND GLUTAMIC-ACID RESIDUES

The interaction of different species variants of cytochrome c and myog lobin, as well as hen egg white lysozyme, with the hard Lewis metal io ns Al3+, Ca2+, Fe3+, and Yb3+ and the borderline metal ion Cu2+, immob ilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investiga ted over the range pH 5.5-8.0. With appropriately chosen buffer and me tal ion conditions, these proteins can be bound to the immobilized M(n +)-IDA adsorbents via negatively charged amino acid residues accessibl e on the protein surface, For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe3-IDA adsorbent, while the other proteins also showed affinity toward i mmobilized Fe3+-IDA adsorbents when buffers containing 30 mM of imidaz ole were used, These studies document that protein selectivity can be achieved with hard-metal-ion immobilized metal ion affinity chromatogr aphy (IMAC) systems through the interaction of surface-exposed asparti c and glutamic acid residues on the protein with the immobilized M(n+) -IDA complex, These investigations have also documented that the so-ca lled soft or borderline immobilized metal ions such as the Cu2+-IDA ad sorbent can also interact with surface-accessible aspartic and glutami c acid residues in a protein-dependent manner. A relationship is evide nt between the number and extent of clustering of the surface-accessib le aspartic and glutamic acid residues and protein selectivity with th ese IMAC systems, The use of elution buffers which contain organic com pound modifiers which replicate the carboxyl group moieties of these a mino acids on the surface of proteins is also described.