New fluorogenic triacylglycerol analogs as substrates for the determination and chiral discrimination of lipase activities

Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings

“…We obtained a specific activity on this substrate of about 5 mIUиmg Ϫ1 for the pure HPL/colipase complex. Our results confirm the data by Duque et al (18) and show that the presence of a single pyrene residue present in the position of one acyl chain from a TAG can dramatically decrease the HPL specific activity in comparison with a non-chemically modified TAG. Owing to the great sensitivity of the fluorescent TAGs containing quenched pyrene, along with the very low specific activity of HPL on this substrate, the effective detection limit is 2 g of HPL (18).…”

“…The sensitivity of this interesting quenched pyrene-based assay for lipase detection is, however, offset by the very poor rate of hydrolysis of the corresponding TAG, as compared to non-chemically modified TAGs (i.e., parinaric acid-containing TAGs). Using the data published by Duque et al (18), we calculated that the specific activity of HPL on the quenched pyrene-modified TAG was about 3 mIUиmg Ϫ1 , versus 150 IUиmg Ϫ1 measured by the same authors on tributyrin. For comparison, we also used a pyrenemodified TAG (with a decanoyl-pyrene in sn-1 position) to assay HPL/colipase in the standard buffer (see Material and Methods).…”

“…The assay involving the use of a TAG containing one acyl chain with a fluorescent pyrene residue linked to the position and another adjacent acyl chain containing a trinitrophenylamine residue as an intramolecular fluorescence quencher (18,19) is a convenient assay for chiral discrimination using purified lipases. The limit of detection of the above fluorophore is highly satisfactory; it can be used to detect amounts as low as one pmole of free fatty acid, which is about a hundred times more sensitive than the parinaric acid detection.…”

“…This intramolecularly quenched TAG containing acyl-pyrene can therefore be used in a continuous fluorescent assay (19). Unfortunately, as can be seen from the data reported in Duque et al (18), this kind of chemically modified TAG is poorly hydrolyzed by lipases, probably for steric reasons. The pyrene group was also used as a fluorophore in continuous assays of lecithin:cholesterol acyltransferase (20), cholesteryl ester hydrolase (21), phospholipase C (22), and phospholipases A (23,24).…”

“…However, this sensitive assay requires the isolation of the released pyrene-fatty acid. A quencher residue (trinitrophenylamine residue) was introduced by Duque et al (18) as a means of lowering the basal fluorescence of this TAG containing acyl-pyrene (1-O-hexadecyl-2-pyrenedecanoyl-3-trinitrophenylaminododecanoylsn -glycerol and its enantiomer). This intramolecularly quenched TAG containing acyl-pyrene can therefore be used in a continuous fluorescent assay (19).…”