Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn²⁺and Ca²⁺in cells

Abstract

Fluorescent dyes are widely used in the detection of labile (free or exchangeable) Zn2+ and Ca2+ in living cells. However, their specificity over other cations and selectivity for detection of labile vs. protein-bound metal in cells remains unclear. We characterized these important properties for commonly used Zn2+ and Ca2+ dyes in a cellular environment. By tracing the fluorescence emission signal along with UV-Vis and size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) in tandem, we demonstrated that among the dyes used for Zn2+, Zinpyr-1 fluoresces in the low molecular mass (LMM) region containing labile Zn2+, but also fluoresces in different molecular mass regions where zinc ion is detected. However, FluoZin™-3 AM, Newport Green™ DCF and Zinquin ethyl ester display weak fluorescence, lack of metal specificity and respond strongly in the high molecular mass (HMM) region. Four Ca2+ dyes were studied in an unperturbed cellular environment, and two of these were tested for binding behavior under an intracellular Ca2+ release stimulus. A majority of Ca2+ was in the labile form as tested by SEC-ICP-MS, but the fluorescence from Calcium Green-1™ AM, Oregon Green® 488 BAPTA-1, Fura red™ AM and Fluo-4 NW dyes in cells did not correspond to free Ca2+ detection. Instead, the dyes showed non-specific fluorescence in the mid- and high-molecular mass regions containing Zn, Fe and Cu. Proteomic analysis of one of the commonly seen fluorescing regions showed the possibility for some dyes to recognize Zn and Cu bound to metallothionein-2. These studies indicate that Zn2+ and Ca2+ binding dyes manifest fluorescence responses that are not unique to recognition of labile metals and bind other metals, leading to suboptimal specificity and selectivity.

Zinc Modulates Endotoxin-Induced Human Macrophage Inflammation through ZIP8 Induction and C/EBPβ Inhibition

“…Therefore, these cations have the potential to modulate the impact of zinc on macrophage immune function through competitive uptake and cell signaling [44, 45]. Although, Zinpyr-1 is highly specific for labile zinc it may be limited in its ability to distinguish between particular divalent cations under certain circumstances [46]. The cumulative impact of ZIP8 on macrophage inflammation in the presence of multiple substrates will require further study.…”

Impact of Cadmium on Intracellular Zinc Levels in HepG2 Cells: Quantitative Evaluations and Molecular Effects

“…In fact, the main pool of intracellular zinc is bound to a vast population of metalloproteins, referred to as the zinc proteome of the cells, or its excess is stored in zinc-containing vesicles or organelles [ 30 ]. In this respect, it should be noted that Zinquin not only binds free zinc (reaction ( 1 )), but can also access Zn-protein with open coordination sites to form fluorescent ternary adducts (reaction ( 2 )), or chelating Zn 2+ from the proteome (reaction ( 3 )) [ 31 , 32 ]: However, most of the zinc bound to proteins is unreactive toward Zinquin. The term “labile” will be used in the following to indicate all of the Zn(II) sensed by Zinquin.…”

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

“…A recent paper also reported the association of NPG with proteomic fractions after gel filtration. 28 …”