New fluorogenic triacylglycerol analogs as substrates for the determination and chiral discrimination of lipase activities.
A new type of fluorogenic and isomerically pure 1(3)-O-alkyl-2,3 (3,2)-diacyl glycerols was synthesized that can be used as substrate for the determination of lipase activities. These compounds contain a fluorescent pyrene acyl chain and, as a potent quencher of pyrene fluorescence, a trinitrophenylamino acyl residue. In their intact form, the fluorogens show only low fluorescence intensity. Upon lipase-induced or chemical hydrolysis of the substrates, however, the fluorophore and quencher separate from each other. This leads to a gradual increase in pyrene fluorescence, reflecting the time-dependent progress of lipolysis and, under substrate saturation conditions, lipase activity. This lipase assay is continuous and does not require separation of substrate and reaction products. Short- and long-chain homologues as well as optical isomers of the fluorogenic alkyldiacyl glycerols were hydrolyzed by pancreatic lipase, hepatic lipase, and lipo-protein lipase at highly different rates depending on the substrate or enzyme preparation and source (e.g., postheparin plasma or cultured cells). It is proposed that a useful set of enantiomeric and/or homologous substrates in combination with appropriate reaction media might be applied to the selective determination of a lipase in a mixture of lipases, e.g., hepatic and lipoprotein lipase in PHP, for medical diagnostics.
Duque
M
M
Department of Biochemistry and Food Chemistry, SFB-Biokatalyse, Technische Universität Graz, Austria.
Graupner
M
M
Stütz
H
H
Wicher
I
I
Zechner
R
R
Paltauf
F
F
Hermetter
A
A
D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006868:Hydrolysis; D066298:In Vitro Techniques; D008049:Lipase / Q000032:analysis / Q000097:blood / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D010179:Pancreas / Q000201:enzymology; D051381:Rats; D013237:Stereoisomerism; D013379:Substrate Specificity; D013552:Swine; D014280:Triglycerides / Q000138:chemical synthesis / Q000737:chemistry*
D005456:Fluorescent Dyes; D014280:Triglycerides; D008049:Lipase; D008071:Lipoprotein Lipase