Newport Green (NPG) is a widely used fluorescent sensor for imaging intracellular Zn2+. The increase of fluorescence intensity upon its exposure has been interpreted as its imaging of labile or weakly bound Zn2+ at the cellular level. However, the slow hydrolysis of its non-fluorescent ester form to fluorescent acid inside LLC-PK1 cells � only about 30% hydrolysis in half an hour � may confuse any increase of fluorescence due to the formation of Zn2+-NPG. Indeed, addition of TPEN, a powerful membrane permeable Zn2+ chelator, made no impact on the fluorescence.  Another peculiarity of the reaction of cells with NPG(ester) was that much of the NPG(acid) generated upon hydrolysis of NPG (ester) was found in the extracellular medium � an observation that implies the efflux of NPG(acid) from the cells. Moreover, studies in our laboratory indicated that the affinity of Newport Green for Zn2+ (Kd ~ 1 �M) was too small for it to bind Zn2+ in the presence of proteome or glutathione ligands. This observation leads to the hypothesis that NPG is unlikely to bind Zn2+ released from intracellular stores by physiological or pathological reactions. Newport Green's weak ability to bind intracellular labile Zn2+ has also been verified by two experiments that examined Zn2+ release from proteome with nitric oxide donors � diethylamine NONOate (DEA-NO) and N-ethylmaleimide (NEM). In both experiments, Zn2+ was released but became bound to proteome in preference to NPG. Upon separating the reaction mixture of proteome and NPG � both ester and acid forms � with Centricon filtration system, the majority of the fluorescence was retained by the filter along with the proteome suggesting the non-specific interaction of NPG with proteome. Therefore, all these experiments lead us to the conclusion that Newport Green is not a very good candidate as a sensor for imaging intracellular Zn2+. Supported by GM-85114 and ES-04184.

How does Newport Green act as a fluorescent sensor for weakly bound cellular Zn<sup>2+</sup>?

248th National Meeting (2014)

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How does Newport Green act as a fluorescent sensor for weakly bound cellular Zn2+?

How does Newport Green act as a fluorescent sensor for weakly bound cellular Zn²⁺?