title: The separation of pancreatic procarboxypeptidases by high-performance liquid chromatography and chromatofocusing.
abstract: Different experimental conditions and chromatographic supports have been selected for the most efficient and rapid purification of procarboxypeptidases from porcine and human pancreas by different high-performance liquid chromatography (HPLC) variants (anion exchange, reversed phase and gel filtration). Anion-exchange chromatography was found to be the most capable and permitted the isolation, in a single step, of three different porcine procarboxypeptidases (2A + 1B forms) and five different human procarboxypeptidases (2B + 3A forms) in a native and pure state from whole pancreas extracts. Other pancreatic proproteases are also cleanly isolated in the same step. Reversed-phase chromatography under mild conditions separated porcine or human procarboxypeptidases A from other pancreatic proteins in a very short time but was unable further to subfractionate the same proteins. The sequential use of gel filtration (or anion-exchange) and reversed-phase HPLC chromatography permitted, in a simple way, the isolation and dissociation of the strongly bound components of the binary complexes between procarboxypeptidases A and proproteinase E in either porcine or human pancreas extracts. Chromatofocusing on a fast protein liquid chromatographic support was also found to be a very efficient technique, showing a slightly lower capability to separate procarboxypeptidases than anion-exchange HPLC though in a much shorter time and in larger quantities.
authors.affiliation: Departament de Bioquímica i Biología Molecular, Universitat Autònoma de Barcelona, Spain.
authors.forename: F J
authors.initials: FJ
authors.lastname: Burgos
authors.forename: R
authors.initials: R
authors.lastname: Pascual
authors.forename: J
authors.initials: J
authors.lastname: Vendrell
authors.forename: C M
authors.initials: CM
authors.lastname: Cuchillo
authors.forename: F X
authors.initials: FX
authors.lastname: Avilés
mesh_terms: D000818:Animals; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000302:isolation & purification*; D043422:Carboxypeptidases A; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D010179:Pancreas / Q000201:enzymology*; D013552:Swine
chemical_list: D004792:Enzyme Precursors; D009097:Multienzyme Complexes; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D043424:Carboxypeptidase B