Abstract
Newport Green (NPG) is a recognized sensor of cellular Zn(2+) that displays fluorescence enhancement upon binding to Zn(2+). Because of its modest affinity for Zn(2+), the extent of its capacity to bind cellular Zn(2+) is unclear. The present study investigated the range of reactivity of NPG(ESTER) with cells, isolated (Zn)-proteome, and model Zn-proteins. The sensor accumulated in pig kidney LLC-PK1 cells and was slowly (>40 min) hydrolyzed to the fluorescent, acid form, NPG(ACID). The powerful, cell permeant Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethane-1,2-diamine (TPEN) failed to quench the growing fluorescence emission, indicating that Zn-NPG(ACID) had not formed and NPG-Zn-protein adduct species probably were not present. Furthermore, NPG(ACID) did not bind to Zn-carbonic anhydrase or Zn-alcohol dehydrogenase, two proteins that form adducts with some other sensors. Strikingly, most of the NPG(ACID) that had been converted from NPG(ESTER) was detected in the extracellular medium not the cells. As a result, after cells were incubated with NPG(ESTER) and then Zn-pyrithione to raise the internal concentration of mobile Zn(2+), Zn-NPG(ACID) was only observed in the external medium. Residual cellular NPG(ACID) was unable to bind extra intracellular Zn(2+) delivered by pyrithione. Proteome isolated from the sonicated cell supernatant was also unreactive with NPG(ACID). Titration of proteome or glutathione with Zn(2+) in the presence of NPG(ACID) revealed that NPG(ACID) only weakly competes for mobile Zn(2+) in the presence of these cellular components. In addition, when proteomic Zn(2+) was released by a nitric oxide donor or N-ethyl-maleimide, little Zn(2+) was detected by NPG(ACID). However, exposure to nitric oxide independently enhanced the fluorescence properties of NPG(ACID). Thus, the biochemical properties of NPG related to cellular Zn(2+) chelation deepen the question of how it functions as a Zn(2+) sensor.