4.8 Article

Selective and Ratiometric Fluorescent Trapping and Quantification of Protein Vicinal Dithiols and in Situ Dynamic Tracing in Living Cells

Journal

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 136, Issue 40, Pages 14237-14244

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/ja5079656

Keywords

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Funding

  1. National Natural Science Foundation of China [21476077, 21236002, 21373138, 21302125]
  2. National Basic Research Program of China (973 Program) [2013CB733700, 2010CB126100]
  3. National High Technology Research and Development Program of China (863 Program) [2011AA10A207]
  4. Shanghai Pujiang Program
  5. Doctoral Fund of Ministry of Education of China [20133127120005]
  6. Program for Shanghai Sci. & Tech. Committee [13ZR1458800]
  7. Fundamental Research Funds for the Central Universities
  8. Program for Cultivation of Young Teacher of Shanghai University

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Protein vicinal dithiols play fundamental roles in intracellular redox homeostasis due to their involvement in protein synthesis and function through the reversible vicinal dithiol oxidation to disulfide. To provide quantitative information about the global distribution and dynamic changes of protein vicinal dithiols in living cells, we have designed and synthesized a ratiometric fluorescent probe (VTAF) for trapping of vicinal dithiol-containing proteins (VDPs) in living cells. VTAF exhibits a ratiometric fluorescence signal upon single excitation, which enables self-calibration of the fluorescence signal and quantification of endogenous vicinal dithiols of VDPs. Its potential for in situ dynamic tracing of changes of protein vicinal dithiols under different cellular redox conditions was exemplified. VTAF facilitated the direct observation of subcellular distribution of endogenous VDPs via ratiometric fluorescence imaging and colocalization assay. And the results suggested that there are abundant VDPs in mitochondria. Moreover, some redox-sensitive VDPs are also present on cell surface which can respond to redox stimulus. This ratiometric fluorescence technique presents an important extension to previous fluorescence intensity-based probes for trapping and quantifying protein vicinal dithiols in living cells, as well as its visible dynamic tracing of VDPs.

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