Phospholipase A2 assay using an intramolecularly quenched pyrene-labeled phospholipid analog as a substrate

Abstract

A phospholipid analog 1-palmitoyl-2–6(pyren-1-yl)hexanoyl- sn-glycero-3-phospho- N-(trinitrophenyl)aminoethanol (PPHTE) in which pyrene fluorescence is intramolecularly quenched by the trinitrophenyl group was used as a substrate for pancreatic phospholipase A2. Upon phospholipase A2 catalyzed hydrolysis of this molecule pyrene monomer fluorescence emission intensity increased as a result of the transfer of the pyrene fatty acid to the aqueous phase. Optimal conditions for phospholipase A2 hydrolysis of PPHTE were similar to those observed earlier for other pyrenephospholipids (T. Thuren, J. A. Virtanen, R. Verger, and P. K. J. Kinnunen (1987) Biochim. Biophys. Acta 917, 411–417) . Although differential scanning calorimetry revealed no thermal phase transitions for PPHTE between +5 and +60°C the Arrhenius plot of the enzymatic hydrolysis of the lipid showed a discontinuity at 30°C. The molecular origin of this discontinuity remains at present unknown. To study the effects of dimyristoylphosphatidylcholine (DMPC) phase transition at 23.9°C on phospholipase A2 reaction PPHTE was mixed with DMPC in a molar ratio of 1:200 in small unilamellar vesicles. The hydrolysis of DMPC-PPHTE vesicles was measured by following the increase in pyrene monomer fluorescence emission due to phospholipase A2 action on PPHTE. Below the phase transition of DMPC the enzymatic reaction exhibited a hyperbolic behavior. At the transition as well as at slightly higher temperatures a lag period was observed. The longest lag period was approximately 20 min. Above 26°C no lag time could be observed. However, the reaction rates were slower than below the phase transition temperature. After the lag period maximal reaction rates were observed at the transition temperature. These results are consistent with results from studies using pure DMPC as substrate for phospholipase A2 and indicate that the trinitrophenyl-labeled pyrenephosphatidylethanolamine did not cause any major perturbation in phospholipase A2-DMPC interaction. Accordingly, these results suggest that PPHTE can be used as a probe to monitor the enzymatic degradation of nonlabeled phosphatidylcholines.