Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Metallomics. 2014 May;6(5):1034-42. doi: 10.1039/c3mt00305a.
Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable. We recently developed a variant of human apocarbonic anhydrase II for sensing metal ions that exhibits 25-fold better selectivity for Cu(II) over Zn(II) than the wild type protein, enabling us to accurately measure Cu(II) in the presence of ordinary cellular (picomolar) concentrations of free zinc. We inserted a fluorescent labeled Cu(II)-specific variant of human apocarbonic anhydrase into PC-12 cells and found that the levels are indeed extremely low (in the femtomolar range). We imaged the free Cu(II) levels in living cells by means of frequency-domain fluorescence lifetime microscopy. Implications of this finding are discussed.
铜是一种必需的微量元素，在许多人类酶中起着关键作用，因此铜缺乏或铜转运的遗传缺陷会导致严重或致命的疾病。Rae 等人曾著名地预测，细胞质中游离铜离子的水平极低，通常低到无法观察到。我们最近开发了一种用于感测金属离子的人脱碳酸酐酶 II 的变体，与野生型蛋白相比，该变体对 Cu(II)的选择性高 25 倍，超过了 Zn(II)，使我们能够在存在普通细胞（皮摩尔级）浓度的游离锌的情况下准确测量 Cu(II)。我们将荧光标记的 Cu(II)特异性人脱碳酸酐酶 II 插入 PC-12 细胞中，发现水平确实极低（在飞摩尔范围内）。我们通过频域荧光寿命显微镜对活细胞中的游离 Cu(II)水平进行了成像。讨论了这一发现的意义。
