High sensitivity, quantitative measurements of polyphosphate using a new DAPI-Based approach
High sensitivity, quantitative measurements of polyphosphate using a new DAPI-Based approach
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DOI：
10.1007/s10895-008-0315-4
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发表时间：
2008-09-01
影响因子：
2.7
通讯作者：
Pavlov, Evgeny
中科院分区：
文献类型：
--
作者：
Aschar-Sobbi, Roozbeh;Abramov, Andrey Y.;Pavlov, Evgeny
聚磷酸盐(PolyP)是原核生物和真核生物体内重要的代谢产物和信号分子。DAPI(4‘，6-二氨基-2-苯基吲哚)是一种广泛使用的DNA荧光标记，也与聚磷酸盐相互作用。Poly-P与DAPI的结合，使其峰值发射波长从475 nm移动到525 nm(激发波长为360 nm)，从而使DAPI能够用于体外和活体聚磷菌中PolyP的检测。这种方法依赖于荧光发射的漂移的检测，仅允许使用DAPI定性检测在微克/毫升范围内的相对高浓度的聚-P。在这里，我们报告了DAPI-Poly-P络合物的长波激发(>；=400 nm)提供了显著提高PolyP检测的灵敏度。在415 nm处激发，DAPI-Poly-P络合物的荧光可以在更高的波长(550 Nm)下被检测到，其最低浓度为25 ng/ml。在该波长下，游离DAPI和DAPI-DNA的荧光发射最小，这使得DAPI-PolyP信号具有高度的特异性，并且基本上独立于DNA的存在。此外，我们还使用该方法来测量多聚磷水解酶和多聚磷酸酶的活性，并从培养神经元的线粒体区显示出类似的信号。
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 mu g/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.