Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.
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).
Hemdan
E S
ES
Institute of Biochemistry, University of Uppsala, Sweden.
Zhao
Y J
YJ
Sulkowski
E
E
Porath
J
J
D002147:Calmodulin / Q000032:analysis; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D003035:Cobalt; D003300:Copper; D003574:Cytochrome c Group / Q000032:analysis; D006639:Histidine / Q000032:analysis*; D008670:Metals; D009113:Muramidase / Q000032:analysis; D009211:Myoglobin / Q000032:analysis; D009532:Nickel; D011506:Proteins / Q000032:analysis*; D013879:Thioredoxins / Q000032:analysis; D014452:Ubiquitins / Q000032:analysis; D015032:Zinc
D002147:Calmodulin; D002413:Cations, Divalent; D003574:Cytochrome c Group; D008670:Metals; D009211:Myoglobin; D011506:Proteins; D014452:Ubiquitins; D003035:Cobalt; D006639:Histidine; D013879:Thioredoxins; D003300:Copper; D009532:Nickel; D009113:Muramidase; D015032:Zinc