Zinc Probe Research Articles

Zinc attenuates monocrotaline-induced pulmonary hypertension in rats through upregulation of A20

Pulmonary hypertension (PH) is characterized by excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), in which inflammatory signaling caused by activation of the NF-κB pathway plays an important role. A20 is an important negative regulator of the NF-κB pathway, and zinc promotes the expression of A20 and exerts a protective effect against various diseases (e.g. COVID19) by inhibiting the inflammatory signaling. The role of A20 and intracellular zinc signaling in PH has been explored, but the extracellular zinc signaling is not well understood, and whether zinc has protective effects on PH is still elusive. Using inductively coupled plasma mass spectrometry (ICP-MS), we studied the alteration of trace elements during the progression of monocrotaline (MCT)-induced PH and found that serum zinc concentration was decreased with the onset of PH accompanied by abnormalities of other three elements, including copper, chromium, and magnesium. Zinc chloride injection with the dosage of 5 mg/kg intraperitoneally partially corrected this abnormality and inhibited the progression of PH. Zinc supplementation induced the expression of A20 in lung tissue and reduce the inflammatory responses. In vitro, zinc supplementation time-dependently upregulated the expression of A20 in PASMCs, therefore correcting the excessive proliferation and migration of cells caused by hypoxia. Using genetically encoded-FRET based zinc probe, we found that these effects of zinc ions are not achieved by entering cells, but most likely by activating cell surface zinc receptor (ZnR/GPR39). These results provide the first evidence of the effectiveness of zinc supplementation in the treatment of PH.

Phytochelatins Bind Zn(II) with Micro- to Picomolar Affinities without the Formation of Binuclear Complexes, Exhibiting Zinc Buffering and Muffling Rather than Storing Functions.

Phytochelatins (PCs) are poly-Cys peptides containing a repeating γ-Glu-Cys motif synthesized in plants, algae, certain fungi, and worms by PC synthase from reduced glutathione. It has been shown that an excess of toxic metal ions induces their biosynthesis and that they are responsible for the detoxification process. Little is known about their participation in essential metal binding under nontoxic, basal conditions under which PC synthase is active. This study presents spectroscopic and thermodynamic interactions with the PC2-PC5 series, mainly focusing on the relations between Zn(II) complex stability and cellular Zn(II) availability. The investigations employed mass spectrometry, UV-vis spectroscopy, potentiometry, competition assays with zinc probes, and isothermal titration calorimetry (ITC). All peptides form ZnL complexes, while ZnL2 was found only for PC2, containing two to four sulfur donors in the coordination sphere. Binuclear species typical of Cd(II)-PC complexes are not formed in the case of Zn(II). Results demonstrate that the affinity for Zn(II) increases linearly from PC2 to PC4, ranging from micro- to low-picomolar. Further elongation does not significantly increase the stability. Stability elevation is driven mainly by entropic factors related to the chelate effect and conformational restriction rather than enthalpic factors related to the increasing number of sulfur donors. The affinity of the investigated PCs falls within the range of exchangeable Zn(II) concentrations (hundreds of pM) observed in plants, supporting for the first time a role of PCs both in buffering and in muffling cytosolic Zn(II) concentrations under normal conditions, not exposed to zinc excess, where short PCs have been identified in numerous studies. Furthermore, we found that Cd(II)-PC complexes demonstrate significantly higher metal capacities due to the formation of polynuclear species, which are lacking for Zn(II), supporting the role of PCs in Cd(II) storage (detoxification) and Zn(II) buffering and muffling. Our results on phytochelatins' coordination chemistry and thermodynamics are important for zinc biology and understanding the molecular basis of cadmium toxicity, leaving room for future studies.

Zip14 ablation modulates HDAC activity through zinc transport in intestinal epithelial cells

Zinc is known for its modulation of proinflammatory responses, in part by targeting nuclear factor kappa B (NF-ĸβ), signal transducer and activator of transcription 3 (STAT3) and expression of specific cytokines, including IFNγ. However, the molecular mechanism underlying Zn-induced cellular response to inflammation remains largely unknown. Our studies, using isolated mouse intestinal epithelial cells (IECs) and mouse intestinal organoids, both derived from Zip14 (Slc39a14) knockout mice, as well as intestinal-specific Zip14 knockout mice, with corresponding control genotypes, were used as models to evaluate the molecular basis for zinc-related inflammatory response in IECs, particularly a potential role for epigenetic modifications. Using chromatin immunoprecipitation (ChIP) assays employing chromatin from IECs, NF-ĸβ was found a major regulator of the Zip14 gene, at steady state. Compared to transcription factors (TFs) STAT3, ATF4 and ATF6, the involvement of NF-ĸβ supports the responsiveness of Zip14 expression with proinflammatory conditions. Similarly, more NF-ĸβ binding was found for iNOS, Cldn-2 and Cldn-8 promoters; all are genes that upregulate in IECs with ablation of Zip14. In contrast, markedly less NF-ĸβ was bound to the promoter of the defense gene IL-18 and C ldn-1; both of which were downregulated with Zip14 ablation. Differential gene expression with Zip14 ablation, as measured by RNA-seq, was limited to ~ 1% of total gene targets, at a false discovery rate of ± 2. RNA from control and Zip14 knockout IECs, intestinal organoids derived from those mice, and an IEC cell line treated with siRNA for Zip14, confirmed the differential expression profiles via qPCR. IECs from the models used here consistently show reduced histone deacetylase (HDAC) activity when Zip14 expression is limited. Moreover, that reduction in activity is concurrent with reduced zinc uptake as measured fluorometrically with the zinc probe, FluoZin-3. Additionally, histone acetylation is greater in IECs when Zip14 is deleted. Treatment of IECs with HDAC inhibitors support a link between Zip14 ablation and HDAC activity. Many HDACs are zinc-metalloenzymes and activity is inhibited with zinc chelation. We propose that deletion of Zip14 lessens HDAC activity, allowing for greater TF occupancy for promoters of specific genes. In summary, we demonstrate that histone acetylation is involved in a zinc-induced anti-inflammatory response in intestinal epithelial cells that is regulated by HDAC activity. Our study shows an important connection between histone modification and the pathological process of zinc-related intestinal inflammation and provides a mechanism for zinc-mediated epigenetic regulation in IECs. Supported by NIH grant DK 094244 and Boston Family Endowment Funds of the University of Florida Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

From methodological limitations to the function of metallothioneins - A guide to approaches for determining weak, moderate, and tight affinity zinc sites.

Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participation in zinc and copper homeostasis. Ever since their discovery, MTs have been investigated in terms of metal-binding affinity. The initial concept of seven Zn(II) ions (Zn7MT) bound with the same, undifferentiated low picomolar affinity in the α and β domains prevailed for many years and derived from spectroscopic studies. The application of fluorescent zinc probes has changed the perception of MTs, showing that they function in nano- to subnanomolar free zinc concentrations due to the presence of tight, moderate, and weak binding sites. The discovery of Zn(II)-depleted MTs in many tissues and determination of cellular free Zn(II) concentrations with differentiated zinc affinity sites revealed the critical importance of partially saturated Zn4-6MTs species in cellular zinc buffering in a wide pico- to nanomolar range of free Zn(II) concentrations. Until today, there is no clear agreement on the presence of differentiated or only tight zinc sites. Here, we present a series of spectroscopic, mass spectrometry-based, and enzymatic competition experiments which reveal how weak, moderate, or high affinity ligands interact with human MT2, with special attention to the determination of Zn(II) affinities. The results show that the simplification of the stability model is the major reason for determining significantly different stability data that obscured the actual MTs function. Therefore, we emphasize that different metal affinities are the single most reason for their presumed function, which changed over the years from tight binding and, thus, storage to one that is highly dynamic.

Intracellular Zinc Trafficking during Crotalus atrox Venom Wound Development.

In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with Crotalus atrox (CA venom) snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of CA venom. HUVEC monolayers stimulated with 10 µg/mL CA venom for 3 h displayed cellular retraction, which coincided with 53.0 ± 6.5 percent viability. In contrast, venom concentrations of 100 µg/mL produced a complete disruption of cellular adherence and viability decreased to 36.6 ± 1.0. The zinc probe Fluozin-3AM was used to detect intracellular zinc in non-stimulated controls, HUVEC stimulated with 10 µg/mL CA venom or HUVEC preincubated with TPEN for 2 h then stimulated with 10 µg/mL CA venom. Fluorescent intensity analysis returned values of 1434.3 ± 197.4 for CA venom demonstrating an increase of about two orders of magnitude in labile zinc compared to non-stimulated controls. Endothelial response to CA venom induced a 96.1 ± 3.0- and 4.4 ± 0.41-fold increase in metallothionein 1X (MT1X) and metallothionein 2A (MT2A) gene expression. Zinc chelation during CA venom stimulation significantly increased cell viability, suggesting that the maintenance of zinc homeostasis during envenomation injury improves cell survival.

A Versatile Molecular Probe of Napthalimide-Derivative for Zn(II) Sensor: A Mini-Review

In recent years, many more chemists have been focused on fluorescent probe research, and significant progresses, which has been made in terms of design and use. This review begins with a brief overview of the most prevalent chemosensor design concepts, followed by a discussion of the photophysical characteristics of the 1,8-naphthalimide structure, such as great photostability, high fluorescence quantum yield and ease of modification which has been used in Zn2+ cation sensing to date. The contributions of the fluorescent probes built on the 1,8-naphthalimide based platform in the field of chemical sensing, biological sensing, pharmaceutical chemistry, environment and food safety in the last 10 years are then systematically introduced by discussing the probe’s fluorescence behavior in the corresponding recognition process. Simultaneously, we intend to build an overall review on fluorescent chemical sensor that will be able to play larger and more interesting role in the future. HIGHLIGHTS Napthalimide-based fluorescence molecular probes for zinc(II) detections are described Various napthalimide derivatives are described based on their potent applications Fluorescence chemosensors are described GRAPHICAL ABSTRACT

Development and Characterization of a Red Fluorescent Protein-Based Sensor RZnP1 for the Detection of Cytosolic Zn2.

Fluorescent sensors have been developed to record Zn2+ dynamics and measure Zn2+ concentrations within the cell. Most previous efforts on developing single-wavelength sensors are focused on green sensors. Here, we engineer a genetically encoded, single red fluorescent protein-based Zn2+ sensor, Red Zinc Probe (RZnP1), which can detect intracellular concentrations of Zn2+. RZnP1 demonstrates a sensitive response to cytosolic Zn2+ (Kd = 438 pM), decent brightness (quantum yield (QY) = 0.15), good in situ dynamic range (Fmax/Fmin = 4.0), and specificity for Zn2+ over other biologically relevant metal cations. RZnP1 offers a way to image Zn2+ with multiple intracellular ions in tandem. We demonstrate the simultaneous recording of Zn2+ and Ca2+ using RZnP1 alongside the Ca2+ sensor GCaMP5G in HeLa cells. We also use RZnP1 with mito-GZnP2, a green fluorescent protein (GFP)-based mitochondrial Zn2+ sensor, to track Zn2+ dynamics in the cytosol and mitochondria concurrently in rat primary neuron culture. Our work not only expands the toolbox of Zn2+ sensors but also demonstrates techniques for imaging Zn2+ dynamics along with other cations and between multiple subcellular compartments simultaneously.

Zinc dynamics regulate early ovarian follicle development

Zinc fluctuations regulate key steps in late oocyte and preimplantation embryo development; however, roles for zinc in preceding stages in early ovarian follicle development, when cooperative interactions exist between the oocyte and somatic cells, are unknown. To understand the roles of zinc during early follicle development, we applied single cell X-ray fluorescence microscopy, a radioactive zinc tracer, and a labile zinc probe to measure zinc in individual mouse oocytes and associated somatic cells within early follicles. Here, we report a significant stage-specific increase and compartmental redistribution in oocyte zinc content upon the initiation of early follicle growth. The increase in zinc correlates with the increased expression of specific zinc transporters, including two that are essential in oocyte maturation. While oocytes in follicles exhibit high tolerance to pronounced changes in zinc availability, somatic survival and proliferation are significantly more sensitive to zinc chelation or supplementation. Finally, transcriptomic, proteomic, and zinc loading analyses reveal enrichment of zinc targets in the ubiquitination pathway. Overall, these results demonstrate that distinct cell type-specific zinc regulations are required for follicle growth and indicate that physiological fluctuation in the localization and availability of this inorganic cofactor has fundamental functions in early gamete development.

Abstract 2454: Imaging pancreatic ductal adenocarcinoma using a zinc-sensitive MRI contrast agent: A novel method to detect early-stage PDAC lesions

Abstract Background: Zinc content and its flux from the exocrine pancreas are both novel candidates for molecular imaging of pancreatic ductal adenocarcinoma (PDAC) due to the markedly dysregulated homeostasis in PDAC lesions. Here, we introduce the use of a Gd-based zinc probe (GdL) to monitor stimulated exocrine function in the healthy and PDAC bearing mouse pancreas by MRI. Methods: Male adult mice were implanted with syngeneic PDAC cells into the pancreas and allowed to form PDAC for 1 and 2 weeks. Healthy mice were used as controls. Mice were fasted overnight and imaged in a 4.7 T scanner. Two 2D T1-weighted gradient echo scans were obtained as baseline (TE/TR = 2.9/125 ms, Avg = 4). Mice then received either 1) 0.07mmol/kg GdL (i.v.) plus 10 ug/kg caerulein (i.p.), 2) 0.07mmol/kg GdL (i.v.) plus Saline (i.p.), or 3) 0.01 mmol/kg Gd-DOTA (i.v.). Immediately following injections, serial 2D T1-weighted gradient echo scans were obtained for 25 minutes. PDAC mice receiving saline were considered as negative controls. Change in contrast to-noise ratio (CNR) of tumor and pancreas was quantified and reported as a function of time. Tumor and pancreas tissue were then fixed, and histological analysis was done to characterize the expression of zinc transporters in healthy pancreas and PDAC tissues. Results: Secretagogue-stimulated zinc secretion (SSZS) MRI of the exocrine pancreas relies on evoking zinc release from intracellular stores from acinar and ductal cells. After i.p. injection of the secretagogue caerulein in fasted healthy animals, we observed only a small enhancement in the pancreas. However, both groups of animals carrying orthotopically implanted PDAC cells (1-wk and 2-wks) responded in hyper-secretory fashion. Negative controls (saline group) did not show significant pancreatic or tumor enhancement. Importantly, the effect of caerulein in PDAC animals and the increase in overall pancreatic enhancement were only found in PDAC tumor bearing animals. Dysregulation of zinc trafficking was confirmed by immunohistochemistry staining for the zinc import transporters ZIP3 and ZIP4. These transporters were upregulated only in PDAC-bearing pancreas and the positive staining was concentrated mostly in the tumor parenchyma and gradually dissipated in the surrounding pancreatic tissue. Conclusions: Zinc flux is markedly upregulated in PDAC. By stimulating the natural zinc secretion from exocrine pancreatic cells using an exogenous secretagogue, caerulein, we can non-invasively monitor early malignant transformations in pancreatic parenchyma and tumor cells by MRI with a Gd-based zinc sensitive probe. Citation Format: Mozhdeh Sojoodi, Ian Ramsay, Eric Gale, Stephen C. Barrett, Motaz Qadan, Peter Caravan, Kenneth K. Tanabe, Veronica Clavijo Jordan. Imaging pancreatic ductal adenocarcinoma using a zinc-sensitive MRI contrast agent: A novel method to detect early-stage PDAC lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2454.

A sensitive zinc probe operating via enhancement of excited-state intramolecular charge transfer.

Novel highly sensitive fluorescent probes for zinc cations based on the diketopyrrolopyrrole scaffold were designed and synthesized. Large bathochromic shifts (≈80 nm) of fluorescence are observed when the Zn2+-recognition unit (di-(2-picolyl)amine) is bridged with the fluorophore possessing an additional pyridine unit able to participate in the coordination process. This effect originates from the dipolar architecture and the increasing electron-withdrawing properties of the diketopyrrolopyrrole core upon addition of the cation. The new, greenish-yellow emitting probes, which operate via modulation of intramolecular charge transfer, are very sensitive to the presence of Zn2+. Introduction of a morpholine unit in the diketopyrrolopyrrole structure induces a selective six-fold increase of the emission intensity upon zinc coordination. Importantly, the presence of other divalent biologically relevant metal cations has negligible effects and typically even at a 100-fold higher concentration of Mg2+/Zn2+, the effect is comparable. Computational studies rationalize the strong bathochromic shift upon Zn2+-complexation. Decorating the probes with the triphenylphosphonium cation and morpholine unit enables selective localization in the mitochondria and the lysosome of cardiac H9C2 cells, respectively.

Innentitelbild: Red‐ and Far‐Red‐Emitting Zinc Probes with Minimal Phototoxicity for Multiplexed Recording of Orchestrated Insulin Secretion (Angew. Chem. 49/2021)

Die Insulinsekretion aus β-Zellen kann nun zusammen mit dem Kalziumsignal und den Organellen aufgezeichnet werden. In ihrem Forschungsartikel auf S. 26050 beschreiben Zhixing Chen und Mitarbeiter die Entwicklung einer neuen Klasse von rot-emittierenden Zn2+-Sonden mit minimaler Phototoxizität. Die neuen Sonden sind für die 4D-, Langzeit- und Mehrfarb-Aufzeichnung der Zn2+/Insulin-Cosekretion in β-Zellen und Inselzellen zugeschnitten und sind geeignet, die räumlich-zeitliche Regulierung der Insel-Endokrinologie zu identifizieren.

Inside Cover: Red‐ and Far‐Red‐Emitting Zinc Probes with Minimal Phototoxicity for Multiplexed Recording of Orchestrated Insulin Secretion (Angew. Chem. Int. Ed. 49/2021)

Insulin secretion from β-cells in intact islets can now be recorded together with calcium signal and organelles. In their Research Article on page 25846, Zhixing Chen and co-workers developed a new class of red- and far-red-emitting Zn2+ probes with minimal phototoxicity. Tailored for 4D, long-term, and multiple-color recording of Zn2+/insulin co-secretion in β-cells and islets, the new probes promise to unveil the spatial-temporal regulation of islet endocrinology.

Red‐ and Far‐Red‐Emitting Zinc Probes with Minimal Phototoxicity for Multiplexed Recording of Orchestrated Insulin Secretion

AbstractZinc biology, featuring intertwining signaling networks and critical importance to human health, witnesses exciting opportunities in the big data era of physiology. Here, we report a class of red‐ and far‐red‐emitting Zn2+ probes with Kd values ranging from 190 nM to 74 μM, which are particularly suitable for real‐time monitoring the high concentration of Zn2+ co‐released with insulin during vesicular secretory events. Compared to the prototypical rhodamine‐based Zn2+ probes, the new class exploits morpholino auxochromes which eliminates phototoxicity during long‐term live recording of isolated islets. A Si‐rhodamine‐based Zn2+ probe with high turn‐on ratio (>100), whose synthesis was enabled by a new route featuring late‐stage N‐alkylation, allowed simultaneous recording of Ca2+ influx, mitochondrial signal, and insulin secretion in isolated mouse islets. The time‐lapse multicolor fluorescence movies and their analysis, enabled by red‐shifted Zn2+ and other orthogonal physiological probes, highlight the potential impact of biocompatible fluorophores on the fields of islet endocrinology and system biology.

Red- and Far-Red-Emitting Zinc Probes with Minimal Phototoxicity for Multiplexed Recording of Orchestrated Insulin Secretion.

Zinc biology, featuring intertwining signaling networks and critical importance to human health, witnesses exciting opportunities in the big data era of physiology. Here, we report a class of red- and far-red-emitting Zn2+ probes with Kd values ranging from 190 nM to 74 μM, which are particularly suitable for real-time monitoring the high concentration of Zn2+ co-released with insulin during vesicular secretory events. Compared to the prototypical rhodamine-based Zn2+ probes, the new class exploits morpholino auxochromes which eliminates phototoxicity during long-term live recording of isolated islets. A Si-rhodamine-based Zn2+ probe with high turn-on ratio (>100), whose synthesis was enabled by a new route featuring late-stage N-alkylation, allowed simultaneous recording of Ca2+ influx, mitochondrial signal, and insulin secretion in isolated mouse islets. The time-lapse multicolor fluorescence movies and their analysis, enabled by red-shifted Zn2+ and other orthogonal physiological probes, highlight the potential impact of biocompatible fluorophores on the fields of islet endocrinology and system biology.

Тhiacalix[4]arene phosphonate C-800 as a novel fluorescent probe for zinc in living cells

Тhiacalix[4]arene phosphonate C-800 as a novel fluorescent probe for zinc in living cells

Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sites.

Here, using human metallothionein (MT2) as an example, we describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity utilizing iodoacetamide (IAM) and N-ethylmaleimide reagents. We concluded that an SN2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissociation. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addition reaction after separation by reverse-phase C18 at acidic pH. Finally, we evaluated the efficiency of the method by mapping metal-binding sites of Zn7–xMT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resolution. The methodology presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-containing proteins.

Deep-Red-Fluorescent Zinc Probe with a Membrane-Targeting Cholesterol Unit.

Organelle-targeting fluorescence probes are valuable because they can provide spatiotemporal information about the trafficking of analytes of interest. The spatiotemporal resolution can be improved by using low-energy emission signals because they are barely contaminated by autofluorescence noises. In this study, we designed and synthesized a deep-red-fluorescent zinc probe (JJ) with a membrane-targeting cholesterol unit. This zinc probe consists of a boron-azadipyrromethene (aza-BODIPY) fluorophore and a zinc receptor that is tethered to a tri(ethylene glycol)-cholesterol chain. In aqueous solutions buffered to pH 7.4, JJ exhibits weak fluorescence with a peak wavelength of 663 nm upon excitation at 622 nm. The addition of ZnCl2 elicits an approximately 5-fold enhancement of the fluorescence emission with a fluorescence dynamic range of 141000. Our electrochemical and picosecond transient photoluminescence investigations indicate that the fluorescence turn-on response is due to the zinc-induced abrogation of the formation of a nonemissive intramolecularly charge-separated species, which occurs with a driving force of 0.98 eV. The fluorescence zinc response was found to be fully reversible and to be unaffected by pH changes or the presence of biological metal ions. These properties are due to tight zinc binding with a dissociation constant of 4 pM. JJ was found to be nontoxic to HeLa cells up to submicromolar concentrations, which enables cellular imaging. Colocalization experiments were performed with organelle-specific stains and revealed that JJ is rapidly internalized into intracellular organelles, including lysosomes and endoplasmic reticula. Unexpectedly, probe internalization was found to permeabilize the cell membrane, which facilitates the influx of exogens such as zinc ions. Such permeabilization does not arise for a control probe without the tri(ethylene glycol)-cholesterol chain (JJC). Our results show that the membrane-targeting cholesterol unit can disrupt membrane integrity.

A novel reversible fluorescent probe for Zinc(II) ion and bioimaging in living cells

ABSTRACT A simple Schiff-base fluorescent probe (L) was synthesised for selective detection of Zn2+ ion in CH3CN/HEPES solution (1/1, 10.0 μM, pH = 7.0). The probe L showed highly fluorescence enhancement towards Zn2+ over other metal ions with obvious fluorescence colour change from faint yellow to blue, which should be attributed to the formation of a 1:2 ligand-metal complex resulting in the inhibition of intramolecular charge transfer (ICT) and the chelation-enhanced fluorescence (CHEF) process. The bonding mechanism was confirmed by UV-vis, fluorescence, HRMS, 1 H NMR experiments and theoretical calculation. Reversibility of the probe in its binding with Zn2+ ion was established in the presence of EDTA. Furthermore, the fluorescence imaging indicated that probe L could be used to effectively identify Zn2+ in living cells.

Ternary Zn(II) Complexes of Fluorescent Zinc Probes Zinpyr-1 and Zinbo-5 with the Low Molecular Weight Component of Exchangeable Cellular Zinc Pool.

The intracellular exchangeable Zn(II) is usually measured with synthetic fluorescent zinc sensors. 4',5'-Bis[bis(2-pyridylmethyl)aminomethyl]-2',7'-dichlorofluorescein (Zinpyr-1) is a sensor containing the fluorescein platform and a duplicated chelating unit. Its advantages include brightness and a relatively high affinity for Zn(II), Kd = 0.7 nM. 2-(4,5-Dimethoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole (Zinbo-5) is a member of a growing family of ratiometric synthetic Zn(II) probes, offering a possibility to determine Zn(II) concentration independently of the sensor concentration. Cells, however, contain high, millimolar or nearly millimolar concentrations of low molecular weight ligands (LMWLs) capable of binding Zn(II) ions. Previously, we demonstrated that such LMWLs can perturb the performance of some fluorescent zinc sensors by competition and formation of ternary Zn(sensor) (LMWL) complexes. Here we tested Zinpyr-1 and Zinbo-5 in this respect. Despite structural differences, both sensors formed such ternary complexes. We determined their stability constants CKtern and performed numerical simulations of Zn(II) distributions at physiological concentrations of selected LMWLs. Glutamic acid was found to provide the strongest ternary complexes with either of the studied sensors. Zn(Zinpyr-1)(Glu) was an absolutely dominant Zn(II)/Zinpyr-1 species (more than 96% of the exchangeable Zn(II)), and Zn(Zinbo-5)(Glu) was the most abundant one (more than 40%) in these simulations. Our results indicate that under cellular conditions these sensors are able to report Zn(II) complexed to LMWLs rather than free Zn2+ ions. On the other hand, the specific affinity of Zn(Zinpyr-1) and Zn(Zinbo-5) for Glu creates interesting opportunities for determining glutamic acid in biological samples.

Interrogating Intracellular Zinc Chemistry with a Long Stokes Shift Zinc Probe ZincBY-4.

Previous work has shown that fluctuations in zinc content and subcellular localization play key roles in regulating cell cycle progression; however, a deep mechanistic understanding requires the determination of when, where, and how labile zinc pools are concentrated into or released from stores. Labile zinc ions can be difficult to detect with probes that require hydrolysis of toxic protecting groups or application at high concentrations that negatively impact cell function. We previously reported a BODIPY-based zinc probe, ZincBY-1, that can be used at working concentrations that are 20-200-fold lower than concentrations employed with other probes. To better understand how zinc pools can be visualized at such low probe concentrations, we modulated the photophysical properties via changes at the 5-position of the BODIPY core. One of these, ZincBY-4, exhibits an order of magnitude higher affinity for zinc, an 8-fold increase in brightness in response to zinc, and a 100 nm Stokes shift within cells. The larger Stokes shift of ZincBY-4 presents a unique opportunity for simultaneous imaging with GFP or fluorescein sensors upon single excitation. Finally, by creating a proxy for the cellular environment in spectrometer experiments, we show that the ZincBY series are highly effective at 50 nM because they can pass membranes and accumulate in regions of high zinc concentration within a cell. These features of the ZincBY probe class have widespread applications in imaging and for understanding the regulatory roles of zinc fluxes in live cells.