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.
Hortin
G L
GL
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110.
Gibson
B L
BL
D002021:Buffers; D002268:Carboxypeptidases / Q000302:isolation & purification*; D002614:Chelating Agents; D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids / Q000378:metabolism; D012685:Sepharose; D015032:Zinc
D002021:Buffers; D002614:Chelating Agents; D007098:Imino Acids; D012685:Sepharose; D002268:Carboxypeptidases; D010450:Endopeptidases; D015032:Zinc; C008109:iminodiacetic acid