Jump to main content
Jump to site search

Issue 2, 2016
Previous Article Next Article

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

Author affiliations

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 NPGESTER 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, NPGACID. 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–NPGACID had not formed and NPG–Zn-protein adduct species probably were not present. Furthermore, NPGACID did not bind to Zn-carbonic anhydrase or Zn-alcohol dehydrogenase, two proteins that form adducts with some other sensors. Strikingly, most of the NPGACID that had been converted from NPGESTER was detected in the extracellular medium not the cells. As a result, after cells were incubated with NPGESTER and then Zn-pyrithione to raise the internal concentration of mobile Zn2+, Zn–NPGACID was only observed in the external medium. Residual cellular NPGACID was unable to bind extra intracellular Zn2+ delivered by pyrithione. Proteome isolated from the sonicated cell supernatant was also unreactive with NPGACID. Titration of proteome or glutathione with Zn2+ in the presence of NPGACID revealed that NPGACID 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 NPGACID. However, exposure to nitric oxide independently enhanced the fluorescence properties of NPGACID. Thus, the biochemical properties of NPG related to cellular Zn2+ chelation deepen the question of how it functions as a Zn2+ sensor.

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

Back to tab navigation
Please wait while Download options loads

Supplementary files

Publication details

The article was received on 22 Jun 2015, accepted on 25 Nov 2015 and first published on 25 Nov 2015


Article type: Paper
DOI: 10.1039/C5MT00167F
Citation: Metallomics, 2016,8, 201-210
  • Open access: Creative Commons BY license
  •   Request permissions

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

    M. R. Karim and D. H. Petering, Metallomics, 2016, 8, 201
    DOI: 10.1039/C5MT00167F

    This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material.

    Reproduced material should be attributed as follows:

    • For reproduction of material from NJC:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
    • For reproduction of material from PCCP:
      [Original citation] - Published by the PCCP Owner Societies.
    • For reproduction of material from PPS:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
    • For reproduction of material from all other RSC journals:
      [Original citation] - Published by The Royal Society of Chemistry.

    Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.

Search articles by author