Metal chelate affinity chromatography of Zn2+-inhibited protein Tyr(P) phosphatases.
Metal chelate affinity chromatography using Zn2+-iminodiacetate agarose is shown to provide quantitative recoveries of Zn2+-inhibited protein Tyr(P) phosphatases. To elute adsorbed enzymes from immobilized Zn2+ three methods were compared: (1) removal of Zn2+ with chelators such as EDTA, (2) introduction of ligands to compete with enzyme for Zn2+ and (3) lowering pH to protonate sidechains in the enzyme that serve as ligands to Zn2+. Results show highest yields but poor purification for method 1, high purification but poor yields of active enzyme for method 3. It is concluded that gradients of competing Zn2+ ligands, such as imidazole, provide the best strategy for the purification of enzymes with retention of activity using metal chelate affinity chromatography.
Shriner
C L
CL
Section of Biochemistry, Brown University, Providence, RI 02912.
Brautigan
D L
DL
D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids; D007700:Kinetics; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D017027:Protein Tyrosine Phosphatases; D012685:Sepharose / Q000031:analogs & derivatives; D015032:Zinc / Q000494:pharmacology*
D007098:Imino Acids; C042660:zinc(II)-iminodiacetate agarose; D012685:Sepharose; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D015032:Zinc