Biophysical Studies of a Synthetic Mimic of the Apoptosis-Detecting Protein Annexin V

A Zn-dipicolylamine coordination compound is shown to sense the presence of anionic phospholipids in a membrane bilayer. The sensor contains two dipicolylamine subunits attached to an anthracene scaffold, which exhibits a maximum absorbance at 380 nm, and undergoes an enhancement in fluorescence intensity when exposed to membranes enriched in phosphatidylserine. For these reasons, the compound is referred to as PSS-380 (Phosphatidylserine Sensor, 380 nm). The fluorescence emission of PSS-380 is enhanced up to tenfold by the presence of vesicles containing the anionic phospholipids phosphatidylserine, phosphatidylglycerol, or phosphatidic acid. No enhancement in fluorescence is observed upon exposure to vesicles containing only zwitterionic phosphatidylcholine, or exposure to monodispersed (non-aggregated) anionic phospholipids. The sensing effect is cooperative; not only does association to the vesicles increase if the vesicles have raised levels of anionic phospholipid, but the maximum fluorescence at sensor saturation is also enhanced. It appears that sensing is triggered by the three-component self-assembly of sensor, Zn, and the anionic membrane surface, which leads to diminished photo-induced electron transfer (PET) quenching. The utility of PSS380 in flow cytometry and fluorescence microscopy is demonstrated by using the molecule to detect the appearance of phosphatidylserine on the plasma membrane surface of various cell lines. Thus, PSS-380 can identify apoptotic cells in the same way as the commonly used protein reagent annexin V.