A fluorescence assay for assessing chelation of intracellular iron in a membrane model system and in mammalian cells
A fluorescence assay for assessing chelation of intracellular iron in a membrane model system and in mammalian cells
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DOI：
10.1006/abio.1996.0032
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发表时间：
1996-01-15
影响因子：
2.9
通讯作者：
Breuer, W
中科院分区：
文献类型：
--
作者：
Cabantchik, ZI;Glickstein, H;Breuer, W
铁螯合剂是铁代谢生化研究和铁超载疾病治疗的重要工具。它们的作用方式包括进入细胞和清除细胞内金属，其中包括络合和排出络合物。铁是一种金属，在细胞中以不同的化学形式和不同的隔间出现。直接受螯合剂影响的金属形式是最不稳定的低分子量类型，存在于细胞质中，被称为“螯合铁”。“这种形式被认为与细胞中存在的几种隔离形式处于动态平衡状态，包括铁反应蛋白。我们最近介绍了一种荧光方法来评估细胞的可螯合铁池，基于荧光钙黄蛋白被金属离子猝灭（Breuer等，J. Biol.）。化学。中文信息学报，1995,27,24209-24215)。在这项工作中，我们采用了动态评估螯合剂在清除细胞铁的功效的方法。在实验1中，红细胞幽灵被用作细胞膜模型。钙黄蛋白的游离酸（非渗透）形式通过封装（裂解和重新密封）加载到鬼影中，其荧光通过添加渗透铁（II）来淬灭。在鬼影中加入螯合剂，使钙黄蛋白中的铁被去除，从而恢复荧光，这与它们对鬼影的渗透和铁的结合亲和力相称。在实验2中，人类K562红白血病细胞通过其渗透性和可切割的乙酰氧基甲基形式装载钙黄蛋白。细胞内钙黄蛋白荧光的一部分在原位被内源性细胞铁淬灭。加入螯合剂后获得的脱冷速率为细胞内金属的清除提供了一种衡量标准，这一过程与试验1中一样，取决于螯合剂的渗透和对铁的结合亲和力。这两种方法为评估候选螯合剂结构在耗尽细胞铁池中的效果提供了方便的手段。它们也可能适用于其他金属的螯合，如Co(II), Ni（II）和Cu(II)，以及任何细胞系统或膜囊泡。(C) 1996学术出版社，Inc.
Iron chelators are important tools in biochemical studies of iron metabolism and in the therapy of iron overload diseases. Their mode of action is comprised of entry into cells and scavenging intracellular metal, which includes complexation and egress of the complex. Iron is a metal which appears in the cells in various chemical forms and in different compartments. The form of the metal directly affected by the chelators is the most labile, low-molecular-weight type, which is present in the cytosol and is known as the ''chelatable iron.'' This form is thought to be in dynamic equilibrium with several sequestered forms present in the cell, including the iron-responsive proteins. We recently introduced a fluorescent method for assessing the chelatable iron pool of cells, based on the quenching of the fluorescent calcein by metal ions (Breuer et al., J. Biol. Chem., 1995, 270, 24209-24215). In this work we adapted the method for dynamic assessment of chelator efficacy in scavenging iron from cells. In assay 1, red blood cells ghosts are used as a cell membrane model. The free-acid (impermeant) form of calcein is loaded into ghosts by encapsulation (lysis and resealing) and its fluorescence is quenched by addition of permeant iron(II). Chelators added to ghosts lead to iron removal from calcein and hence to recovery of fluorescence, commensurate with their permeation into ghosts and iron binding affinity. In assay 2, human K562 erythroleukemia cells are loaded with calcein via its permeating and cleavable acetoxymethyl form. A fraction of the intracellular calcein fluorescence is quenched in situ by endogenous cellular iron. The rate of dequenching which is obtained after addition of a chelator provides a measure for the scavenging of the intracellular metal, a process which, as in assay 1, depends on chelator permeation and binding affinity for iron. The two methods provide convenient means for assessing the efficacy of candidate chelator structures in depleting cell iron pools. They are also potentially applicable to chelation of other metals such as Co(II), Ni(II), and Cu(II) and to any cellular system or membrane vesicles. (C) 1996 Academic Press, Inc.