Analysis of Inorganic Polyphosphates by Capillary Gel Electrophoresis

Abstract

This paper describes the development of a method that uses Capillary Gel Electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((Pi)n). Resolution of (Pi)n on the basis of n, the number of residues of dehydrated phosphate, is accomplished by CGE using capillaries filled with solutions of poly(N,N-dimethylacrylamide) (PDMA) and indirect detection by the UV-absorbance of a chromophore, terephthalate, added to the running buffer. The method is capable of resolving peaks representing (Pi)n with n up to ~70; preparation and use of authentic standards enables the identification of peaks for (Pi)n with n = 1 - 10. The main advantages of this method over previously reported methods for analyzing mixtures of (Pi)n (e.g., gel electrophoresis, CGE using polyacrylamide-filled capillaries) are its resolution, convenience, and reproducibility; gel-filled capillaries are easily regenerated by pumping in fresh, low-viscosity solutions of PDMA. The resolution is comparable to that of ion-exchange chromatography and detection of (Pi)n by suppressed conductivity. The method is useful for analyzing (Pi)n generated by the dehydration of Pi at low temperature (125 - 140 °C) with urea, in a reaction that may have been important in prebiotic chemistry. The method should also be useful for characterizing mixtures of other anionic, oligomeric or polymeric species without an intrinsic chromophore (e.g., sulfated polysaccharides, oligomeric phospho-diesters).

Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity

“…Because the accumulation of these compounds inside vacuoles mostly depends on proton antiporters (Russnak et al, 2001;Tomitori et al, 2001;Shimazu et al, 2005;Brohée et al, 2010), the electrochemical potential could support polyP translocation by the import of these ligands into vacuoles. Protonation of polyP is unlikely to play a major role here, because the pH of vacuole lumen is 5.5 (Brett et al, 2011), whereas the sole hydroxyl group on each phosphate residue of polyP is strongly acidic (pK a ,1-2) (Lee and Whitesides, 2010). A further hypothesis, which we favor, is that translocation is directly driven by the electrical potential across the vacuolar membrane.…”

Ppn2, a novel Zn2+-dependent polyphosphatase in the acidocalcisome-like yeast vacuole

“…First, the end and middle groups of polyP have different pK a values. While the middle groups are strongly acidic, the pK a1 of the end groups is between 6.5 and 7.2 -close enough to the luminal pH of vacuoles to influence the buffering capacity of the vacuole lumen (Lee and Whitesides, 2010). Shifting the average chain length towards shorter chains can increase the concentration of the end groups and might play a role in controlling vacuolar and cytosolic pH.…”

Methods for the Analysis of Polyphosphate in the Life Sciences

“…Capillary electrophoresis is performed with submillimeter diameter capillaries. The stationary phase can be polyacrylamide, 78 polydimethylsiloxane, 79 or silica gel. 80 PolyP with a chain length of <70 Psubunits can be separated.…”