Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion-chelate adsorbents.
In this investigation, human serum has been used as an example of a crude protein mixture to define the protein binding characteristics and selectivity of several immobilised hard Lewis metal ion affinity chromatographic (IMAC) adsorbents. Specifically, the binding properties of immobilised O-phosphoserine (im-OPS) and 8-hydroxyquinoline (im-8-HQ), with immobilised iminodiacetic acid as a control system, have been investigated in combination with the hard Lewis metal ions, Al3+, Ca2+, Fe3+, Yb3+, and the borderline metal ion, Cu2+, over the pH range pH 5.5 to pH 8.0 with buffers of 0.5 M ionic strength. The same IMAC adsorbents were also investigated for their protein binding capabilities with buffers of an ionic strength of 0.06 M at pH 5.5 and pH 8.0. The binding behaviour of four "marker" proteins, namely transferrin, alpha2-macroglobulin, gammaglobulin and human serum albumin have furthermore been employed to monitor the differences in protein selectivity exhibited by these IMAC systems. The experimental findings confirm that these hard Lewis metal ion IMAC adsorbents function in a "mixed" binding mode with both coordination and electrostatic characteristics evident, depending on the ionic strength and pH of the equilibration or elution buffers. Based on a screening protocol, several members of the im-Mn+-8-HQ and im-Mn+-OPS adsorbent series have been identified with high selectivity for transferrin and alpha2-macroglobulin. These hard Lewis metal ion IMAC adsorbents thus provide attractive alternatives for selective fractionation of human serum proteins.
Zachariou
M
M
Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Hearn
M T
MT
D001798:Blood Proteins / Q000032:analysis*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification; D007502:Iron Chelating Agents / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D009994:Osmolar Concentration; D011485:Protein Binding; D014168:Transferrin / Q000302:isolation & purification; D000511:alpha-Macroglobulins / Q000302:isolation & purification
D001798:Blood Proteins; D007074:Immunoglobulin G; D007502:Iron Chelating Agents; D008670:Metals; D014168:Transferrin; D000511:alpha-Macroglobulins