Lim Mi Hee, Xu Dong, Lippard Stephen J
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, USA.
Nat Chem Biol. 2006 Jul;2(7):375-80. doi: 10.1038/nchembio794. Epub 2006 May 28.
Nitric oxide (NO) serves as a messenger for cellular signaling. To visualize NO in living cells, we synthesized a turn-on fluorescent probe for use in combination with microscopy. Unlike existing fluorescent sensors, the construct--a Cu(II) complex of a fluorescein modified with an appended metal-chelating ligand (FL)--directly and immediately images NO rather than a derivative reactive nitrogen species. Using spectroscopic and mass spectrometric methods, we established that the mechanism of the reaction responsible for the NO-induced fluorescence involves reduction of the complex to Cu(I) with release of the nitrosated ligand, which occurs irreversibly. We detected NO produced by both constitutive and inducible NO synthases (cNOS and iNOS, respectively) in live neurons and macrophages in a concentration- and time-dependent manner by using the Cu(II)-based imaging agent. Both the sensitivity to nanomolar concentrations of NO and the spatiotemporal information provided by this complex demonstrate its value for numerous biological applications.
一氧化氮（NO）作为细胞信号传导的信使。为了在活细胞中可视化NO，我们合成了一种用于显微镜检查的开启型荧光探针。与现有的荧光传感器不同，该构建体——一种用附加的金属螯合配体（FL）修饰的荧光素的铜（II）配合物——直接且即时地成像NO，而不是衍生的活性氮物种。通过光谱和质谱方法，我们确定了导致NO诱导荧光的反应机制涉及配合物还原为Cu（I）并释放亚硝化配体，这一过程不可逆。通过使用基于铜（II）的成像剂，我们以浓度和时间依赖性方式在活神经元和巨噬细胞中检测到组成型和诱导型一氧化氮合酶（分别为cNOS和iNOS）产生的NO。对纳摩尔浓度NO的敏感性以及该配合物提供的时空信息都证明了其在众多生物学应用中的价值。
