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 chromato graphy (IMAC) have been probed using a set of Saccharomyces cerevisiae iso-1-cytochrome c histidine variants constructed by site-directed mu tagenesis. Proteins containing a single accessible histidine exhibit L angmuir-type isotherms with maximum protein binding capacities between 5 and 10% of the maximum copper loading and the capacity of the suppo rt to bind imidazole. A simple model that assumes that the copper site s are densely packed and can be blocked by protein adsorption yields b inding constants for single-histidine proteins that are similar to the binding constant for free imidazole. Proteins containing multiple acc essible histidines do not exhibit simple Langmuir-type behavior; they appear to interact with the support by simultaneous coordination to mo re 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 signi ficantly weaker at low surface concentrations of copper that presumabl y cannot support multiple-site interactions. The protein binding capac ity drops to zero at copper loadings less than one-half the maximum, i ndicating that immobilized iminodiacetic acid ligands are sufficiently close together that two can coordinate a single copper ion, which pre cludes 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.