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A Sensitive, Nonradioactive Assay for Zn(II) Uptake into Metazoan Cells
Christopher E R Richardson1, Elizabeth M Nolan1, Matthew D Shoulders1
1Department of Chemistry , Massachusetts Institute of Technology , 77 Massachusetts Avenue , Cambridge , Massachusetts 02139 , United States.
Abstract:
Sensitive measurements of cellular Zn(II) uptake currently rely on quantitating radioactive emissions from cells treated with 65Zn(II). Here, we describe a straightforward and reliable method employing a stable isotope to sensitively measure Zn(II) uptake by metazoan cells. First, biological medium selectively depleted of natural abundance Zn(II) using A12-resin [Richardson, C. E. R., et al. (2018) J. Am. Chem. Soc. 140, 2413] is restored to physiological levels of Zn(II) by addition of a non-natural Zn(II) isotope distribution comprising 70% 70Zn(II). The resulting 70Zn(II)-enriched medium facilitates quantitation of Zn(II) uptake using inductively coupled plasma-mass spectrometry (ICP-MS). This sensitive and reliable assay assesses Zn(II)-uptake kinetics at early time points and can be used to delineate how chemical and genetic perturbations influence Zn(II) uptake. Further, the use of ICP-MS in a Zn(II)-uptake assay permits simultaneous measurement of multiple metal ion concentrations. We used this capability to show that, across three cell lines, Zn(II) deficiency enhances selectivity for Zn(II) over Cd(II) uptake.
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