The purification of ovine pancreatic lipase that is free of colipase using an improved delipidation method.
A modified procedure for the purification of ovine pancreatic lipase (triacylglycerol acyl-hydrolase, EC3.1.1.3) is described. The method is more rapid and more reproducible than that reported previously and results in a pure lipase preparation, that gives a better yield at the same specific activity, free of colipase and uncontaminated by lipid. The procedure involves the preparation of a lipid-free acetone powder from fresh pancreas without the use of chloroform or butanol as was used in the procedure described earlier. The aqueous purification of the lipase from the delipidated powder is similar to that described earlier, but includes the use of beta-mercaptoethanol and uses salt gradient elution from CM-Sepharose. An assay procedure for lipase is reported involving the extraction of released free fatty acids with chloroform/methanol before titrating with sodium hydroxide. A modification of this assay is used for the determination of colipase. The above assay procedure is compared to the potentiometric method reported previously. Polyacrylamide gel, amino acid composition analysis and N-terminal sequence data for the purified ovine lipase are presented.
Gieseg
S P
SP
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Forrester
I T
IT
Carne
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003089:Colipases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification*; D008623:Mercaptoethanol; D008722:Methods; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry / Q000201:enzymology*
D000596:Amino Acids; D003089:Colipases; D008623:Mercaptoethanol; D008049:Lipase