Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins-thioredoxin, ubiquitin, calmodulin, lysozyme, cytochrome c, and myoglobin on immobilized transition metal ions (Co2+, Ni2+, Cu2+, and Zn2 )-allows establishment of the following facets of the histidyl side chain distribution: (i) either interior or surface; (ii)-when localized on the surface, accessible or unaccessible for coordination; (iii) single or multiple; (iv) when multiple, either distant or vicinal. Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution).The underlying principle of immobilized metal ion affinity chromatography (IMAC) of proteins is the coordination between the electron donor groupings on a protein surface (histidine, tryptophan, cysteine) and chelated (iminodiacetate; IDA) transition metal ions [IDA-M(II)]. This principle of immobilized metal ion affinity (IMA) has been presented by now in some detail (1-4).The practice of IMAC in the purification of proteins has had its empirical phase. There is now a need, from the body of data, to establish somewhat more detailed ground rules that would allow for the use of IMAC in a more predictive manner.Our present experience (4) clearly indicates that histidine functions as the predominant ligand in the IMAC of proteins based on metal ions belonging to the latter part of the first series of transition metal elements (and zinc). Therefore, topography of the protein surface with respect to the location of histidine residues becomes critical for better understanding and exploitation of an IMA event (1, 4-6).A facile probe into the topography of histidyl residues could be quite useful, especially if it could provide information about their surface disposition. This information is required for the rational exploitation of IMAC for protein isolation. In a simple case, one has only to seek information about the surface accessibility of a putative electron donor grouping (7). However, the evaluation of an IMA event in the case of proteins replete in histidine residues may require more comprehensive analysis (8).We now report that IMAC may be exploited as an analytical tool in addition to its use as a protein purification technique (9,10). IMAC can be used to ascertain several facets of the status of a histidyl...
