High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach

Roozbeh Aschar-Sobbi, Andrey Y. Abramov, Catherine Diao, Margaret E. Kargacin, Gary J. Kargacin, Robert J. French, Evgeny Pavlov

Research output: Contribution to journalArticle

Abstract

Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4′,6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the μg/ml range. Here, we report that long-wavelength excitation (≥400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.

Original languageEnglish (US)
Pages (from-to)859-866
Number of pages8
JournalJournal of Fluorescence
Volume18
Issue number5
DOIs
StatePublished - Sep 2008

Keywords

  • DAPI
  • Fluorescence
  • Inorganic phosphate
  • Polyphosphatase
  • Polyphosphate

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Psychology
  • Social Sciences (miscellaneous)
  • Sociology and Political Science
  • Spectroscopy
  • Clinical Biochemistry
  • Law

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  • Cite this

    Aschar-Sobbi, R., Abramov, A. Y., Diao, C., Kargacin, M. E., Kargacin, G. J., French, R. J., & Pavlov, E. (2008). High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach. Journal of Fluorescence, 18(5), 859-866. https://doi.org/10.1007/s10895-008-0315-4