title: Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes.
abstract: We report on the determination of active enzyme components in pure and crude lipases, using fluorescent inhibitors for covalent modification and visualization of the enzymatically active proteins. Lipase-specific compounds are triacylglycerol analogs, namely 1,2(2, 3)-di-O-alkylglyceroalkylphosphonic acid-p-nitrophenyl esters, containing a fluorescent substituent bound to the omega-end of an alkyl chain. Inhibitors derived from single-chain alcohols, such as p-nitrophenyl esters of fluorescent alkyl phosphonates, react with lipases and esterases. The p-nitrophenyl ester bond is susceptible toward nucleophilic attack by the active serine of the lipolytic enzyme. This reaction is stoichiometric, specific, and irreversible. Stable lipid-protein complexes are formed which can be analyzed on the basis of their fluorescent signal. From fluorescence intensity the moles of active serine (enzyme) were accurately determined. A lipase-specific inhibitor was used for the analysis of a commercial lipase preparation from Rhizomucor miehei. After incubation of the enzyme with the fluorescent lipid, a single fluorescence band was observed after SDS-gel electrophoresis, indicating the presence of a single lipase in the crude enzyme material. A linear correlation was obtained between fluorescence intensity and the amount of enzyme. Using a combination of different inhibitors, we were able to discriminate between lipases and esterases.
authors.affiliation: Department of Biochemistry and Food Chemistry, Technische Universität Graz, SFB-Biocatalysis, Petersgasse 12/2, Graz, A-8010, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Scholze
authors.forename: H
authors.initials: H
authors.lastname: Stütz
authors.forename: F
authors.initials: F
authors.lastname: Paltauf
authors.forename: A
authors.initials: A
authors.lastname: Hermetter
mesh_terms: D001665:Binding Sites; D019117:Burkholderia / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008049:Lipase / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D010569:Perylene / Q000031:analogs & derivatives / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry; D020103:Rhizomucor / Q000201:enzymology; D012233:Rhizopus / Q000201:enzymology; D012697:Serine Endopeptidases / Q000032:analysis; D014280:Triglycerides / Q000737:chemistry
chemical_list: C403778:1-O-hexadecyl-2-O-perylenedodecyl-sn-glycero-3-phosphonic acid-(n-hexyl)-4-nitrophenyl ester; C403779:1-O-octyl-2-O-pyrenebutyl-sn-glycero-3-phosphonic acid-(n-hexyl)-4-nitrophenyl ester; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C403782:O-perylenebutylmethylphosphonic acid-4-nitrophenyl ester; D010743:Phospholipids; D011721:Pyrenes; D014280:Triglycerides; D010569:Perylene; D008049:Lipase; D012697:Serine Endopeptidases; C049262:esterase B