Development of a bis-pyrene phospholipid probe for fluorometric detection of phospholipase A₂ inhibition

In this work, we report the design, synthesis, and application of a bis-pyrene phospholipid probe for detection of phospholipase A₂ action through changes in pyrene monomer and excimer fluorescence intensities. Continuous fluorometric assays enabled detection of the activities of multiple PLA₂ enzymes as well as the decrease in catalysis by PLA₂ from honey bee venom caused by the inhibitor p-bromo phenacylbromide. Thin-layer chromatography and mass spectrometry analysis were also used to validate probe hydrolysis by PLA₂. Mass spectrometry data also supported cleavage of the probe by phospholipase C and D enzymes, although changes in fluorescence were not observed in these cases. Nevertheless, the bis-pyrene phospholipid probe developed in this work is effective for detection of PLA₂ enzyme activity through an assay that enables screening for inhibitor development.