Purification of acetate kinase by affinity chromatography.

A one-step procedure using affinity chromatography has been shown to purify to apparent homogeneity acetate kinase from a commercially available preparation and to partially purify the enzyme from a crude, cell-free extract. Since the gel's capacity for enzyme adsorption is controlled by the thermodynamics of ligand-enzyme interaction, maximization of the adsorption isotherm was attempted. Enzyme adsorption decreased logarithmically with increasing ionic strength but increased with increasing concentration of MgCl2. These competing effects caused the net adsorption of enzyme to increase to a maximum and then to decrease as the MgCl2 concentration was raised. The results allow a significant improvement in affinity column performance and have important implications for scale-up procedures.     

Purification of carboxypeptidase B by zinc chelate chromatography

A method is described to purify pancreatic carboxypeptidases B (CPB), removing contaminating endoproteinases that interfere with use of CPB for carboxy-terminal analysis or modification of proteins. The separation uses zinc chelate chromatography and is based on the property that CPB has higher affinity for immobilized zinc ions than do serine proteinases such as trypsin and chymotrypsin, which are abundant endoproteolytic activities in pancreas. CPB preparations are loaded onto a column of iminodiacetic acid-Sepharose that has been saturated with ZnCl2. A step gradient with buffers of decreasing pH is used to elute bound proteins. CPB elutes at a lower pH than do the serine proteinases.     

Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.