Methods for the Analysis of Polyphosphate in the Life Sciences

Abstract

Inorganic polyphosphate (polyP) is the polymer of orthophosphate and can be found in all living organisms. For polyP characterization, one or more of six parameters are of interest: the molecular structure (linear, cyclic, or branched), the concentration, the average chain length, the chain length distribution, the cellular localization, and the cation composition. Here, the merits, limitations, and critical parameters of the state-of-the-art methods for the analysis of the six parameters from the life sciences are discussed. With this contribution, we aim to lower the entry barrier into the analytics of polyP, a molecule with prominent, yet often incompletely understood, contributions to cellular function. See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. b Refers to absorption and fluorescence spectroscopy. Analytical Chemistry pubs.acs.org/ac Feature https://dx.

Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates

“…The amount of polyP was quantified after polyP extraction followed by digestion to phosphate with exopolyphosphatase and subsequent phosphate determination (malachinte green assay) as recently summarized in (Christ et al 2020b).…”

Biotechnological synthesis of water‐soluble food‐grade polyphosphate with Saccharomyces cerevisiae

“…The nucleic acid and protein content in the bio‐polyP were estimated with a NanoDrop (Thermo Fisher Scientific) by unspecific UV absorption measurement. The polyP purity was determined by measuring the polyP concentration with the Christ et al (2020) enzyme assay, and quantifying the cation composition with atomic absorption spectroscopy or optical emission spectroscopy (see Supplementary Information for the calculations). The cyclic polyP content was measured with 31 P NMR (Glonek, Van Wazer, Mudgett, & Myers, 1972; see Supplementary Information for the calculations).…”

“…The polyP structure is classified in linear, cyclic, and branched polyP, whereas microorganisms produce only linear polyP. See Christ, Willbold, and Blank (2020) for a detailed review of the molecular polyP structure. Whereas alkaline earth metal salts of linear polyP are water‐insoluble, alkali metal salts of linear polyP are water‐soluble (Kulaev et al, 2005, p. 10; Van Wazer, 1958, p. 671).…”

Polyanions in Coagulation and Thrombosis: Focus on Polyphosphate and Neutrophils Extracellular Traps

“…As PPK only digests polyP with a chain length of greater than 38 phosphate-subunits the malachite green assay is insensitive for short chain polyP. PolyP can furthermore be visualized by microscopy (75), electrophoresis, chromatography, 32 P-NMR, Fourier transform-infrared (FT-IR), and mass spectrometry (81).…”

“…• Microscopy 13 • Flow cytometry 81 • Flow chamber 113 • ELISA 82 • Western blotting 19 • Sytox Green/PicoGreen staining 12 • DAPI, Hoechst 33342, toluidine blue O, methylene blue, tetracycline, neutral red, malachite green [85][86][87] • Flow cytometry 79,87 • Urea-polyacrylamide gel electrophoresis 84 • Chromatography 84 • 32 P-NMR 84 • Fourier transform-infrared (FT-IR) 84 • Mass spectrometry 84 • Microscopy 84…”