Chang Christopher J, Jaworski Jacek, Nolan Elizabeth M, Sheng Morgan, Lippard Stephen J
Department of Chemistry, Picower Center for Learning and Memory, and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1129-34. doi: 10.1073/pnas.0308079100. Epub 2004 Jan 20.
Zinc is an essential metal ion for human growth and development, the disruption of cellular Zn(2+) homeostasis being implicated in several major disorders including Alzheimer's disease, diabetes, and cancer. The molecular mechanisms of Zn(2+) physiology and pathology are insufficiently understood, however, owing in part to the lack of tools for measuring changes in intracellular Zn(2+) concentrations with high spatial and temporal fidelity. To address this critical need, we have synthesized, characterized, and applied an intracellular fluorescent probe for the ratiometric imaging of Zn(2+) based on a tautomeric seminaphthofluorescein platform. Zin-naphthopyr 1 (ZNP1) affords single-excitation, dual-emission ratiometric detection of intracellular Zn(2+) through Zn(2+)-controlled switching between fluorescein and naphthofluorescein tautomeric forms. The probe features visible excitation and emission profiles, excellent selectivity responses for Zn(2+) over competing Ca(2+) and Mg(2+) ions at intracellular concentrations, a dissociation constant (K(d)) for Zn(2+) of <1 nM, and an 18-fold increase in fluorescence emission intensity ratio (lambda(624)/lambda(528)) upon zinc binding. We demonstrate the value of the ZNP1 platform for biological applications by imaging changes in intracellular [Zn(2+)] in living mammalian cells. Included is the ratiometric detection of endogenous pools of intracellular Zn(2+) after NO-induced release of Zn(2+) from cellular metalloproteins. We anticipate that ZNP1 and related probes should find utility for interrogating the biology of Zn(2+).
锌是人体生长发育所必需的金属离子，细胞内锌离子（Zn(2+)）稳态的破坏与包括阿尔茨海默病、糖尿病和癌症在内的几种主要疾病有关。然而，由于缺乏能够在高空间和时间分辨率下测量细胞内Zn(2+)浓度变化的工具，人们对Zn(2+)生理和病理的分子机制了解不足。为满足这一关键需求，我们基于互变异构半萘荧光素平台合成、表征并应用了一种用于Zn(2+)比率成像的细胞内荧光探针。锌萘并吡咯1（ZNP1）通过Zn(2+)控制的荧光素和萘荧光素互变异构形式之间的切换，实现对细胞内Zn(2+)的单激发、双发射比率检测。该探针具有可见的激发和发射光谱，在细胞内浓度下对Zn(2+)的选择性响应优于竞争性的钙离子（Ca(2+)）和镁离子（Mg(2+)），Zn(2+)的解离常数（K(d)）<1 nM，锌结合后荧光发射强度比（λ(624)/λ(528)）增加18倍。我们通过对活的哺乳动物细胞内[Zn(2+)]变化进行成像，证明了ZNP1平台在生物学应用中的价值。其中包括在一氧化氮（NO）诱导细胞金属蛋白释放Zn(2+)后，对细胞内Zn(2+)内源性池的比率检测。我们预计ZNP1及相关探针将在研究Zn(2+)生物学方面发挥作用。
