title: Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues.
abstract: 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 range pH 5.5-8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized Mn+-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-metal-ion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surface-exposed aspartic and glutamic acid residues on the protein with the immobilized Mn+-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 of clustering of the surface-accessible aspartic and glutamic 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Zachariou
authors.forename: M T
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000032:analysis / Q000737:chemistry*; D002021:Buffers; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods; D003574:Cytochrome c Group / Q000032:analysis / Q000737:chemistry; D004285:Dogs; D005260:Female; D018698:Glutamic Acid / Q000032:analysis / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids; D008670:Metals / Q000737:chemistry; D009113:Muramidase / Q000032:analysis / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012685:Sepharose / Q000031:analogs & derivatives; D012756:Sheep; D013045:Species Specificity; D013499:Surface Properties
chemical_list: D002021:Buffers; D003574:Cytochrome c Group; D007098:Imino Acids; D008670:Metals; D009211:Myoglobin; D011506:Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; C035771:Sepharose CL 4B; D012685:Sepharose; D009113:Muramidase; C008109:iminodiacetic acid