High-performance liquid chromatography of amino acids, peptides and proteins: CXXI. 8-Hydroxyquinoline-metal chelate chromatographic support: an additional mode of selectivity in immobilized-metal affinity chromatography

Abstract

In our associated studies, the binding of proteins in immobilized-metal affinity chromatography has been shown to be independent of surface-exposed histidines and aromatic amino acid residues, when hard metals are used. The present investigation documents the behaviour of iminodiacetic acid (IDA) and 8-hydroxyquinoline (8-HQ) immobilized on Sepharose CL-4B and chelated with detergent (Brij-35), at pH 7.0. the 8-HQ gel had a higher capacity for tuna heart cytochrome c (THCC) when Fe 3+ was immobilized than when Al 3+ or Yb 3+ was used, whilst 8-HQ-Cu 2+ and 8-HQ-Ca 2+ did not bind this protein. The equivalent IDA chelates showed no binding of the protein. The THCC was recovered from the 8-HQ-Fe 3+, -YB 3+ and -Al 3+ supports upon elution with high concentrations of phosphate, glutamate or malonic acids, suggesting that acidic amino acid residues were involved in the binding. Application of molecular graphics procedures reveals that the 8-HQ-metals 3+ chelate represents a new class of coordination geometry for binding to proteins, and hence offers an additional mode of selectivity in immobilized-metal affinity chromatographic separations.