High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach.
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 microg/ml range. Here, we report that long-wavelength excitation (> or = 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.
Aschar-Sobbi
Roozbeh
R
Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada.
Abramov
Andrey Y
AY
Diao
Catherine
C
Kargacin
Margaret E
ME
Kargacin
Gary J
GJ
French
Robert J
RJ
Pavlov
Evgeny
E
D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005470:Fluorometry; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D011122:Polyphosphates / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007211:Indoles; D011122:Polyphosphates; C007293:DAPI