Multipoint binding in metal-affinity chromatography II. Effect of pH and imidazole on chromatographic retention of engineered histidine-containing cytochromes c.

Protein binding in immobilized metal affinity chromatography (IMAC) was studied using a set of Saccharomyces cerevisiae iso-1-cytochrome c variants which differed only in their histidine content and placement. Elution with an imidazole gradient enabled separation of cytochrome c variants based on their histidine multiplicity. Millimolar concentrations of imidazole dramatically decreased protein partitioning to the IMAC support as measured by the chromatographic capacity factors under isocratic conditions. Fitting the partitioning data to the "stoichiometric displacement" model indicates that cytochrome c variants containing from one to four surface histidines each displaced approximately three equivalents of imidazole upon adsorption. Therefore even a protein with a single surface histidine appears to coordinate to multiple copper sites on the IMAC support at neutral pH. The effect of pH on the capacity factors of these variants measured in the absence of imidazole further supports this interpretation. Although the presence of a surface histidine was required for retention at neutral pH, a variant with no surface histidines still partitioned strongly to the IMAC support at higher pH (pH > 7.5). These results indicate the contribution of additional protein-metal-coordinating groups, presumably surface amines, to chromatographic retention in IMAC.