High-performance liquid chromatography of amino acids, peptides and proteins. CXXXI. O-phosphoserine as a new chelating ligand for use with hard Lewis metal ions in the immobilized-metal affinity chromatography of proteins.
Conditions for the immobilization of O-phosphoserine (OPS) to epoxy-activated Sepharose CL-4B are described. The binding behaviour of OPS and iminodiacetic acid (IDA) immobilized onto Sepharose CL-4B, toward the hard Lewis metal ions Al3+, Fe3+, Ca2+ and Yb3+, and Cu2+ ion as a borderline metal ion control, over the pH range pH 4.0 to pH 8.0, was examined. Immobilized OPS shows a stronger affinity for Fe3+ and Al3+ ions but a lower affinity for Cu2+ and Yb3+ ions, compared to immobilized iminodiacetic acid (IDA), over the equilibrating range examined. Immobilized OPS-Mn+ was screened for protein binding using as model proteins tuna heart cytochrome c (THCC), horse myoglobin (HMYO) and hen egg while lysozyme (HEWL) over the pH range 5.5 to 8.0. Immobilized OPS-Fe3+ bound THCC under all the examined equilibrating conditions, bound HMYO between pH 5.5 and pH 7.0 and did not bind HEWL under any condition examined. Immobilized OPS thus presents an additional mode of metal ion and protein selectivity in immobilized-metal affinity chromatography.
Zachariou
M
M
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Traverso
I
I
Hearn
M T
MT
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000302:isolation & purification; D004529:Egg White / Q000032:analysis; D006736:Horses; D007098:Imino Acids; D008024:Ligands; D008670:Metals / Q000737:chemistry*; D009206:Myocardium / Q000201:enzymology; D010768:Phosphoserine / Q000737:chemistry*; D012685:Sepharose / Q000031:analogs & derivatives; D013696:Temperature; D014413:Tuna
D000596:Amino Acids; D002614:Chelating Agents; D003574:Cytochrome c Group; D007098:Imino Acids; D008024:Ligands; D008670:Metals; D010768:Phosphoserine; C035771:Sepharose CL 4B; D012685:Sepharose; C008109:iminodiacetic acid