Comparison of the binding behavior of several histidine-containing proteins with immobilized copper(II) complexes of 1,4,7-triazacyclononane and 1,4-bis(1,4,7-triazacyclononan-1-yl)butane

The protein binding characteristics of the immobilized binucleating chelate system, 1,4-bis(1,4,7-triazacyclononan-1-yl)butane (tacn2butane), complexed with Cu2+ ions have been investigated with hen egg white lysozyme, horse skeletal muscle myoglobin and horse heart cytochrome C, as well as three histidine-rich proteins, serum albumin, transferrin, and α2-macroglobulin, present in partially fractionated human serum. The effects of pH, ionic strength and elution buffers on protein binding have been examined and compared with those of the analogous immobilized mononuclear copper complex of 1,4,7-triazacyclononane (tacn). The Cu2+-tacn2butane system was generally found to exhibit higher protein binding affinities than the Cu2+-tacn system, suggesting that the presence of immobilized binuclear copper(II) species leads to enhanced coordinative interaction with surface-exposed amino acid residues of the studied proteins. However, under some buffer conditions the dependencies of protein binding and elution on pH and ionic strength with these immobilized metal ion affinity chromatographic (IMAC) systems were consistent with electrostatic, hydrophobic and π-bonding interactions playing a significant secondary role in addition to the dominant coordinative interactions. As such, the results indicated that the selectivities were not solely dependent on the histidine content of the protein. In accord with this conclusion, differences in the selectivities of the Cu2+-tacn and Cu2+-tacn2butane adsorbents for serum albumin, transferrin, and α2-macroglobulin were observed depending on the choice of elution buffer. This attribute suggests that additional selectivity features can be realised for the separation of specific proteins with this new class of adsorbent.