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Zinc-buffering capacity of a eukaryotic cell at physiological pZn

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Abstract

In spite of the paramount importance of zinc in biology, dynamic aspects of cellular zinc metabolism remain poorly defined at the molecular level. Investigations with human colon cancer (HT-29) cells establish a total cellular zinc concentration of 264 μM. Remarkably, about 10% of the potential high-affinity zinc-binding sites are not occupied by zinc, resulting in a surplus of 28 μM ligands (average K cd  = 83 pM) that ascertain cellular zinc-buffering capacity and maintain the “free” zinc concentration in proliferating cells at picomolar levels (784 pM, pZn = 9.1). This zinc-buffering capacity allows zinc to fluctuate only with relatively small amplitudes (ΔpZn = 0.3; below 1 nM) without significantly perturbing physiological pZn. Thus, the “free” zinc concentrations in resting and differentiated HT-29 cells are 614 pM and 1.25 nM, respectively. The calculation of these “free” zinc concentrations is based on measurements at different concentrations of the fluorogenic zinc-chelating agent and extrapolation to a zero concentration of the agent. It depends on the state of the cell, its buffering capacity, and the zinc dissociation constant of the chelating agent. Zinc induction of thionein (apometallothionein) ensures a surplus of unbound ligands, increases zinc-buffering capacity and the availability of zinc (ΔpZn = 0.8), but preserves the zinc-buffering capacity of the unoccupied high-affinity zinc-binding sites, perhaps for crucial physiological functions. Jointly, metallothionein and thionein function as the major zinc buffer under conditions of increased cellular zinc.

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Notes

  1. “Free” zinc has been referred to as “freely available,” “labile,” or “rapidly exchangeable” zinc that is readily bound to chelating agents. Each term is an operational definition and has its limitations. For the lack of a better term, “free” zinc is used in this work, albeit with the understanding that the chemical nature of the ligands of ionic zinc is not known. “Rapidly exchangeable” implies certain kinetic mechanisms. Thus, there are pools of thermodynamically tightly bound zinc with considerable “kinetic lability” in exchange reactions. A prime example is metallothionein.

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Acknowledgements

This work was supported by National Institutes of Health Grant GM 065388 to WM. We thank Dr. V.M. Sadagopa Ramanujam, Associate Professor, Department for Preventive Medicine and Community Health, The University of Texas Medical Branch, for metal analyses by atomic absorption spectrophotometry (supported by the Human Nutrition Research Facility) and Drs. Christopher J. Frederickson and Hans-Werner Adolph for discussions.

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Authors and Affiliations

  1. Division of Human Nutrition, Department of Preventive Medicine and Community Health, University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX, 77555, USA

    Artur Krężel & Wolfgang Maret

  2. Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, 77555, USA

    Wolfgang Maret

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  1. Artur Krężel
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  2. Wolfgang Maret
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Correspondence to Wolfgang Maret.

Appendices

Appendix 1

Variation of “free” zinc in the presence of two ligands, where L1 represents the bulk of cellular zinc proteins and L2 represents the fluorescent probe. The simulations demonstrate that under zinc-buffering conditions extrapolation with a linear function (Fig. 2) is permissible. The concentrations of total zinc and probe correspond to those experimentally determined. The buffered system (Fig. 7, panel A) corresponds to the experimental condition of an excess of unbound ligands (292 μM, corresponding to the sum of 264 μM occupied and 28 μM surplus ligands), whereas the unbuffered (264 μM) system (Fig. 7, panel B) corresponds to a fictional condition without additional zinc-buffering capacity. Intermediate conditions with L1 values of 270, 275, 280, 285, 290, and 295 μM are represented in Fig. 7, panel C, curves a–e, respectively.

Fig. 7
figure 7

Relation between the concentration of FluoZin-3 and “free” zinc in a buffered (a) and an unbuffered (b) system, and in systems with different buffering capacity (c)

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Appendix 2

Simulation of the Zincon–zinc titration (Fig. 8) in the presence of intracellular unbound ligands with various affinities for zinc and experimentally determined parameters (Fig. 4).

Fig. 8
figure 8

Zincon–Zn titration as a function of cellular ligands with different dissociation constants: 83 pM (squares), 3.2 nM (triangles), and 10 nM (circles). The dissociation constant (pKd = 4.9) of the Zn-Zincon complex at pH 7.4 and ε620 = 23,200 M−1 cm−1 were from  [17]. Zincon concentration 200 μM; total cellular ligands 24.53 μM; total zinc 22.18 μM; zinc added 0–6.38 μM

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Krężel, A., Maret, W. Zinc-buffering capacity of a eukaryotic cell at physiological pZn. J Biol Inorg Chem 11, 1049–1062 (2006). https://doi.org/10.1007/s00775-006-0150-5

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