Purification of canine post-heparin hepatic lipase.
A method for the purification of canine hepatic lipase from post-heparin hepatic venous blood plasma was developed and found applicable to mixed venous post-heparin plasma. The method employs sequential (NH4)2SO4 fractionation, heparin-Sepharose chromatography at pH 8.8 and, finally, adsorption to antiserum prepared against dog pre-heparin plasma. The lipase was purified 10,000-fold. The specific activity assayed with Intralipid as substrate was 840 mumol free fatty acid h-1 . mg-1. Enzyme recovery was 20%. Upon electrophoresis of the purified lipase in polyacrylamide gel containing SDS, a major protein-staining band with an apparent molecular weight of 60,000 was consistently found. This component accounted for 85-90% of the protein applied to the gel, and by amino acid analysis appeared to be distinct from canine antithrombin III, a protein thought to contaminate hepatic lipase purified by earlier methods.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004285:Dogs; D006493:Heparin; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000097:blood* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D013045:Species Specificity
D000596:Amino Acids; D006493:Heparin; D008049:Lipase