Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Shellaina  J.V. Gordon; Yao Xiao; Amanda  L. Paskavitz; Napoleón Navarro-Tito; Juan  G. Navea; Teresita Padilla-Benavides

doi:10.3791/59519

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Method Article

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

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DOI:

10.3791/59519

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May 16th, 2019

Shellaina J.V. Gordon^*¹ , Yao Xiao^*² , Amanda L. Paskavitz³ , Napoleón Navarro-Tito⁴ , Juan G. Navea² , Teresita Padilla-Benavides¹

¹Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, ²Department of Chemistry, Skidmore College, ³Candiac MR Center, Beth Israel Deaconess Medical Center, ⁴Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero

^* These authors contributed equally

Summary

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Abstract

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson’s and Menkes’ diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Introduction

Transition and heavy metals, such as Zn, Cu, Mn, and Fe, are found naturally in the environment in both nutrients in food and pollutants. All living organisms require different amounts of these micronutrients; however, exposure to high levels is deleterious to organisms. Metal acquisition is mainly through the diet, but metals can also be inhaled or absorbed through the skin¹^,²^,³^,⁴^,⁵. It is important to note that the presence of metals in atmospheric particles is increasing and has been largely associated w....