Gordon Shellaina J V, Xiao Yao, Paskavitz Amanda L, Navarro-Tito Napoleón, Navea Juan G, Padilla-Benavides Teresita
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School.
Department of Chemistry, Skidmore College.
J Vis Exp. 2019 May 16(147). doi: 10.3791/59519.
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.
过渡金属是生物体必需的微量营养素，但在高浓度时会与蛋白质中的生理金属竞争并产生氧化还原应激，从而对细胞有毒害作用。导致金属缺乏或积累的病理状况是多种人类疾病的致病因素。一些例子包括贫血、肠病性肢端皮炎、威尔逊氏病和门克斯病。因此，能够高灵敏度和高精度地测量生物样品中过渡金属的水平和转运，对于促进探索这些元素如何影响正常生理功能和毒性的研究非常重要。例如，锌（Zn）是许多哺乳动物蛋白质中的辅助因子，参与信号传导事件，并且是细胞中的第二信使。过量的锌有毒，会抑制其他金属的吸收，而缺乏锌则会导致各种潜在的致命状况。石墨炉原子吸收光谱法（GF-AAS）为测定各种生物样品中的锌和其他过渡金属浓度提供了一种高度灵敏且有效的方法。通过GF-AAS进行电热原子化，通过雾化少量样品来量化金属，随后使用感兴趣元素的激发波长进行选择性吸收分析。在比尔-朗伯定律的线性范围内，金属对光的吸光度与分析物的浓度成正比。与其他测定锌含量的方法相比，GF-AAS能够高灵敏度地检测蛋白质中以及可能在小细胞内分子中的游离锌和络合锌，且所需样品量小。此外，GF-AAS也比电感耦合等离子体质谱法（ICP-MS）或基于同步加速器的X射线荧光法更容易获得。在本文中，描述了在GF-AAS中对不同培养细胞系进行分析的系统样品制备方法。作为原理验证，比较了增殖细胞和分化细胞的全细胞裂解物和亚细胞组分中这种微量元素的差异。
