Newport Green, a fluorescent sensor of weakly bound cellular Zn2+: competition with proteome for Zn2+

Abstract

Newport Green (NPG) is a recognized sensor of cellular Zn²⁺ that displays fluorescence enhancement upon binding to Zn²⁺. Because of its modest affinity for Zn²⁺, the extent of its capacity to bind cellular Zn²⁺ 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 Zn²⁺ 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 Zn²⁺, Zn–NPG_ACID was only observed in the external medium. Residual cellular NPG_ACID was unable to bind extra intracellular Zn²⁺ delivered by pyrithione. Proteome isolated from the sonicated cell supernatant was also unreactive with NPG_ACID. Titration of proteome or glutathione with Zn²⁺ in the presence of NPG_ACID revealed that NPG_ACID only weakly competes for mobile Zn²⁺ in the presence of these cellular components. In addition, when proteomic Zn²⁺ was released by a nitric oxide donor or N-ethyl-maleimide, little Zn²⁺ 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 Zn²⁺ chelation deepen the question of how it functions as a Zn²⁺ sensor.