ENANTIOMERIC PERYLENE-GLYCEROLIPIDS AS FLUOROGENIC SUBSTRATES FOR A DUAL-WAVELENGTH ASSAY OF LIPASE ACTIVITY AND STEREOSELECTIVITY

A new type of fluorogenic alkyldiacyl glycerols was synthesized and us ed as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain perylene as a fluorophor e and the trinitrophenylamino (TNP) residue as a quencher. Both substi tuents are covalently bound to the omega-ends of the sn-2 and sn-1(3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase ac tivity by the continuous increase in fluorescence intensity which is c aused by dequenching. Using enantiomeric pairs of these compounds, we were able to analyze lipase stereoselectivity depending on the reactio n medium. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labelled with pyrene or perylene as fluorophores, can be u sed for a dual-wavelength ''stereoassay'' of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be determined simultaneo usly, and the difference in the rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase. Hydrolysis rates measu red with perylene-substituted lipids are generally lower than those ob tained with the pyrene analogs. Thus, with a mixture of perylene and p yrene-substituted lipids, we observe a higher apparent stereoselectivi ty of lipases since we measure a combination of stereo- and substrate selectivity. In the presence of albumin, all microbial lipases tested so far exhibit stereopreference for the sn-1 glycerol position. In our assay, the apparent stereoselectivities are highest if in the presenc e of albumin, the sn-1 position carries pyrene and the sn-3 position i s substituted with perylene. The lipase stereoselectivity assay descri bed here requires the simultaneous measurement of the fluorescence int ensities at two different wavelengths in a single cuvette and can thus be carried out using existing and cheap instrumentation that was deve loped for the fluorimetric analysis of Ca++ concentrations. (C) 1996 W iley-Liss, Inc.