Immobilized metal ion affinity electrophoresis: a preliminary report.

The ligand "Sepharose-IDA-Cu(II)" was entrapped into an agarose gel used for affinity electrophoresis. The binding of three closely related proteins, namely alpha-chymotrypsinogen A, alpha-chymotrypsin, and alpha-chymotrypsin inactivated with diisopropyl fluorophosphate (DIFP) to the affinity gel, was investigated. When the protein having affinity for the ligand was run in the presence of small amounts of the ligand, the retention of the protein by the ligand caused "tailing" of the sample. This pattern was changed in the presence of increasing amounts of the ligand, leading to a "rocket" shape due to the stronger binding of the protein to the chelated metal ligand entrapped in the gel. The degree of retardation in the gel with the ligand is an expression of the affinity between the protein and the ligand. The migration distance of alpha-chymotrypsin and alpha-chymotrypsin treated with DIFP at a given concentration of the ligand is linearly related to the protein amount deposited on the gel. The dissociation constant for the tested proteins were calculated from the Bøg-Hansen-Takeo plot. The difference in the affinity strength of these structurally related proteins towards the ligand suggests the involvement of the surface topography of histidine residues on their binding to the ligand.