The use of C₆-NBD-PC for assaying phospholipase A₂-activity: Scope and limitations

Determination of phospholipase A₂ (PLA₂) activity is of special interest in view of the abundance of this enzyme in various organelles of all cells, and its role in many cell functions, especially in eicosanoid production. Assaying PLA₂ activity is therefore of special importance to cell biology. However, it is a complicated and non-trivial task for several reasons including the critical dependence of PLA₂ activity on the physical state of the lipid substrate, the complex kinetics of its action, the low activity of most intercellular and membrane-bound enzymes and the metabolism of the fatty acid products, when applied to intact cell. In recent years the fluorescent analogue of phosphatidylcholine, C₆-NBD-PC, has been used for determination of the activity of soluble and membrane-bound PLA₂. In the present study we evaluate the use of this method for continuous monitoring of PLA₂ activity, based on time-dependent changes in the fluorescence intensity which results from the hydrolysis of C₆-NBD-PC into NBD-caproic acid and lysolecithin. The fluorescence intensity of aggregated C₆-NBD-PC is reduced due to self-quenching which is maximal in systems containing no additional lipids, when NBD-PC forms micelles. In these systems the hydrolysis increase the fluorescence intensity due to de-quenching, since the NBD caproic acid products dissolves in water in the form of monomers. In contrast, in the presence of additional lipids (mixed micelles, membrane vesicles, lipid emulsion particles or lipoproteins), the probe partitions into lipidic compartments where its fluorescence is only partially guenched (if at all) and its quantum yield is much higher. Consequently, the hydrolysis is accompanied by a decrease in fluorescence. The time course of this change is a complex function of the additional lipid concentration(s) and of physical processes which follow the hydrolysis. Due to this complexity, the assay of PLA₂ activity by continuous monitoring of fluorescence is ambiguous. Furthermore, the rate of NBD-PC hydrolysis is very different from that of the 'host' lipid bilayer or monolayer and is less sensitive to the physical state of the lipids. Under various conditions it follows very different kinetics, depending on the ratio of NBD-PC to the host PC. Therefore, it can not be used as a general assay for PLA₂ in lipid-containing systems.