The separation of pancreatic procarboxypeptidases by high-performance liquid chromatography and chromatofocusing.
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.
Burgos
F J
FJ
Departament de Bioquímica i Biología Molecular, Universitat Autònoma de Barcelona, Spain.
Pascual
R
R
Vendrell
J
J
Cuchillo
C M
CM
Avilés
F X
FX
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
D004792:Enzyme Precursors; D009097:Multienzyme Complexes; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D043424:Carboxypeptidase B