Short communicationThe zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium
Introduction
Zinc in tight association with a variety of proteins plays an important role in the life of cells as a structural or catalytic element [1]. Calcium which enters into looser associations with proteins acts as a second messenger molecule, initiating a number of cellular processes when its ‘free’ concentration is elevated intracellularly. There are some indications that zinc may serve a similar role, however, because the free zinc concentration appears to be held at levels a few orders of magnitude below that of calcium, it has been far more difficult to demonstrate this role conclusively [2]. Fluorescent probes have played an invaluable role in demonstrating and visualizing the role of calcium as a second messenger. In the last few years there has been considerable activity in the development of zinc probes [3], with the hope that such indicators may disclose whether free zinc does indeed serve as a cellular messenger.
A number of zinc sensitive fluorescent probes with a variety of different zinc-binding moieties and fluophores have been developed. From a cell biological perspective these indicators can be divided into two categories; those that cross membranes and those that do not [4]. Membrane permeant probes such as TFLZn [5], Zinquin [6], ZnAF-2 [7], ZP1 [8] and ZP4 [9] have proved useful in exploring the zinc-rich vesicles found in a number of cell types. In contrast, membrane impermeant probes can be used to visualize zinc in the extracellular space [10], [11]. Of these the one that has perhaps the firmest credentials as an impermeant probe is FluoZin-3 (registered trademark of Molecular Probes Inc.), which has been successfully used to detect zinc release from pancreatic β-cells [10] and what has been termed the ‘externalization’ of zinc from hippocampal slices [11], as well as measuring the affinity of the metallothionein zinc-binding sites [12]. FluoZin-3, in the form of an acetoxymethyl (AM) ester derivative, has also proved useful in a variety of biological preparations for detecting intracellular zinc elevations [13], [14], [15], [16], [17], [18], [19].
In contrast to the simplicity of defined aqueous media, the complex heterogenous environment within a cell complicates the interpretation of the changes in fluorescence of indicator molecules [4]. Moreover, zinc indicators in the extracellular space have the additional challenge of functioning in a milieu where the concentration of calcium and magnesium are many orders of magnitude higher than zinc. These factors present pitfalls to the investigator attempting to make sense of fluorescent signals emanating from zinc indicators within the confines of biological tissue.
In this communication we present data on the interaction of FluoZin-3 with zinc, in the presence of chelators, calcium and magnesium.
Section snippets
Fluorimetry
Excitation–emission spectra and fluorescent time-courses were determined on a Hitachi F-4500 spectrofluorimeter using a stirred methacrylate cuvette (Fisher Scientific) whose temperature was controlled by a circulating water bath at 26 °C. The monochromator slit widths were set at 5 nm and the data was sampled at 1 Hz. All experiments were performed in a Hepes buffered saline containing (in mM): 140 NaCl, 2.5 KCl and 10 Hepes (pH 7.4) with FluoZin-3 at a concentration of 500 nM unless otherwise
Results
FluoZin-3 exhibits a single excitation peak at 494 nm and an emission peak at 518 nm, neither of which shift on the addition zinc or any other biological transition metals. Little contaminating metal was found in the analysis of a commercial sample of FluoZin-3. As we have shown previously on addition of the probe to a methacrylate cuvette the fluorescence increases as a result of zinc being leached from the cuvette and stir bar, that is accelerated by stirring [20].
During the course of these
Discussion
The high prevailing levels of calcium and magnesium in the extracellular solution surrounding cells provide a challenge to detecting transient elevations of zinc. FluoZin-3 appears to be well matched to this task because of its low affinity for calcium and magnesium. It is worth noting that the first use of FluoZin-3, which was to detect zinc release from pancreatic β-cells, was made in the presence of 2.4 mM Ca and 1.4 mM Mg [10]. We have shown here that paradoxically the provision of low levels
Conflict of interest
Kyle Gee is an employee of Molecular Probes Invitrogen.
Acknowledgement
This work was supported in part by a grant from NINDS (NS47508 to ARK).
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