MULTIPLE-SITE BINDING INTERACTIONS IN METAL-AFFINITY CHROMATOGRAPHY .1. EQUILIBRIUM BINDING OF ENGINEERED HISTIDINE-CONTAINING CYTOCHROME-C

Mechanisms of protein retention in immobilized metal-affinity chromatography (IMAC) have been probed using a set of Saccharomyces cerevisiae iso-1-cytochrome c histidine variants constructed by site-directed mutagenesis. Proteins containing a single accessible histidine exhibit Langmuir-type isotherms with maximum protein binding capacities between 5 and 10% of the maximum copper loading and the capacity of the support to bind imidazole. A simple model that assumes that the copper sites are densely packed and can be blocked by protein adsorption yields binding constants for single-histidine proteins that are similar to the binding constant for free imidazole. Proteins containing multiple accessible histidines do not exhibit simple Langmuir-type behavior; they appear to interact with the support by simultaneous coordination to more than one metal ion, the result of which is to increase the apparent binding affinity by as much as a factor of 1000. The protein binding constant depends on the availability of copper sites: binding is significantly weaker at low surface concentrations of copper that presumably cannot support multiple-site interactions. The protein binding capacity drops to zero at copper loadings less than one-half the maximum, indicating that immobilized iminodiacetic acid ligands are sufficiently close together that two can coordinate a single copper ion, which precludes its interaction with a protein. Protein adsorption via multiple-site coordination has important consequences for the optimization of IMAC separations and the design of new IMAC supports.