Identification of various lipolytic enzymes in crude porcine pancreatic lipase preparations using covalent fluorescent inhibitors.
We developed a specific method for determination and discrimination of lipo-/estero-lytic enzymes in crude lipase preparations. Here we study the composition of commercial porcine pancreatic lipase (PPL), since it is widely used for bioconversions of synthetic and natural substrates. Our method is based on incubation of enzyme samples with fluorescently labeled alkyl- or dialkylglyceryl-phosphonates in an appropriate solvent followed by protein separation by electrophoresis and fluorescence detection with a CCD camera. After incubation with short-chain alkylphosphonate solubilized by taurodeoxycholate, crude PPL preparations showed a very weak band at 50 kDa, which is indicative of low PPL concentrations in these samples. In addition, seven other fluorescent bands were detected. The band at the lowest molecular weight corresponded to alpha-chymotrypsin. Two intensive fluorescent bands were in the molecular weight range of chymotrypsinogen (26 kDa) and four weak bands were in the range 20-24 kDa. Long-chain dialkylglycerophosphonate labeled two protein bands in crude PPL: alpha-chymotrypsin and a very intensive band corresponding to the molecular weight of chymotrypsinogen. Detection of cholesterol esterase (98 kDa) in crude PPL preparations depended on addition of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) to the incubation mix, as demonstrated by spiking with cholesterol esterase. Thus, commercial crude PPL preparations contain a variety of estero-/lipo-lytic enzymes in addition to rather low amounts of active PPL, which should be considered when using crude PPL for bioconversions. Our method can also be used to show whether an isolated esterolytic activity corresponds to a single protein or isoenzymes. Here we confirm by 2D-electrophoretic separation of "pure" PPL that PPL exists as isoenzymes in different glycosylated forms.
Birner-Grünberger
Ruth
R
Department of Biochemistry, Technische Universität Graz, Petersgasse 12/2, A-8010 Graz, Austria.
Scholze
Hubert
H
Faber
Kurt
K
Hermetter
Albin
A
D000818:Animals; D002457:Cell Extracts / Q000032:analysis*; D004586:Electrophoresis / Q000379:methods*; D008049:Lipase / Q000032:analysis* / Q000737:chemistry*; D008066:Lipolysis; D010179:Pancreas / Q000737:chemistry / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013552:Swine
D002457:Cell Extracts; D008049:Lipase