Biophysical studies of a synthetic mimic of the apoptosis‐detecting protein annexin v

Abstract

A Zn2+‐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, Zn2+, and the anionic membrane surface, which leads to diminished photo‐induced electron transfer (PET) quenching. The utility of PSS‐380 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.

Fluorescent Detection of Apoptotic Cells by Using Zinc Coordination Complexes with a Selective Affinity for Membrane Surfaces Enriched with Phosphatidylserine

“…[20] Association of PSS-380 to a PS-rich membrane leads to fluorescence enhancement, due to enhanced binding of Zn 2 + to the DPA units in PSS-380, which decreases photoinduced electron transfer (PET) quenching. [28] In contrast, the PET-quenching pathway is not significant in PSS-480, and control studies with vesicles show that association of PSS-480 with PS-rich membranes does not alter its fluorescence intensity. Similarly, the fluorescence intensity of PSSgreen QD is unchanged upon membrane binding.…”

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

“…Among suggested compounds the most attention was directed towards the synthetic zinc(II)-di-2-picolylamine (Zn 2? -DPA) coordination complexes (Koulov et al 2005). Similarly to Ca 2?…”

“…So, in this case also the selective recognition of apoptotic cells needs a three-component assembly process. In the initially suggested compound, two DPA subunits were attached to an anthracene scaffold absorbing light at 380 nm, and this compound was called PSS-380 (Koulov et al 2005). Two of Zn 2?…”

Imaging and therapeutic applications of zinc(ii)-dipicolylamine molecular probes for anionic biomembranes

“…86 The fluorescence intensity of PSVue-380 was found to increase by 10-fold when titrated with anionic vesicles that contained PS, PG, or phosphatidic acid, but was unchanged when treated with vesicles comprised solely of the zwitterionic PC. 87 Association of the sensor with the membrane surface occurs by a ‘three component’ self-assembly process where the DPA scaffold containing one Zn 2+ ion and a free Zn 2+ ion are attracted to the negatively charged membrane surface and form a bridged complex with the anionic head group of a phospholipid (Scheme 4). The successful targeting of model vesicle systems prompted us to evaluate whether PSVue-380 could operate in more complicated biological environments.…”