Journal of Protein Chemistry

, Volume 14, Issue 6, pp 419–430 | Cite as

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues

  • Michael Zachariou
  • Milton T. W. Hearn


The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al3+, Ca2+, Fe3+, and Yb3+ and the borderline metal ion Cu2+, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the rangepH 5.5–8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized M n +-IDA adsorbents via negatively charged amino acid residues accessible 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 immobilized Fe3+-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metalion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surfaceexposed aspartic and glutamic acid residues on the protein with the immobilized M n +-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu2+-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number and extent of clustering of the surfaceaccessible aspartic and glutamic acid residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.

Key words

Immobilized metal ion affinity chromatography cytochrome c surface accessibility hard metal ion chelates 



iminodiacetic acid


iminodiacetic acid chelated to metal ion


immobilized metal affinity chromatography


dog heart cytochrome c


horse heart cytochrome c, THCC, tuna heart cytochrome c


horse skeletal muscle myoglobin


sheep skeletal muscle myoglobin


hen egg white lysozyme


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Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Michael Zachariou
    • 1
  • Milton T. W. Hearn
    • 1
  1. 1.Centre for Bioprocess Technology, Department of Biochemistry and Molecular BiologyMonash UniversityClaytonAustralia

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