# An Update on Polyphosphate In Vivo Activities
## 2 Biophysical Characteristics of Polyphosphate  
Polyphosphate is an inorganic anionic energy-rich polymer widespread in all living organisms, from bacteria to mammalians [7]. Furthermore, the polymer is also produced synthetically, and synthesised polyP is used in large amounts in an array of applications, e.g., as a water softener, fire extinguisher, or as additives for conservation, pH buffer, and water retention in the food industry [8]. Synthetic polyP molecules are technically produced by condensation of orthophosphoric acid monomers with dehydration under high temperatures. Polymerisation generates energy-rich phosphoanhydrid bonds that form the backbone of linear and branched polymers and supramolecular polyP structures [9]. Thenucleotides GTP and ATP share these high-energy phosphoanhydride bonds with polyP. In nature, enzymatic hydrolysis of the terminal high-energy \(\beta\)-\(\gamma\) and \(\alpha\)-\(\beta\) phosphoanhydride bonds in ATP to ADP and ADP to AMP, respectively, liberates \(\sim\)30 kJ/mol of energy [5]. Corresponding to the hydrolysis of the terminal ATP phosphoanhydride bond, the hydrolysis of P-O-P anhydride bonds in linear polyP similarly generates \(\sim\)30 kJ/mol under standard conditions [10; 11]. The kinetics of the P\({}_{\text{i}}\)-generating hydrolysis reaction is slow. At physiological pH, polyP has a half-life of about 90 min in plasma [12] and several months in aqueous solutions ex vivo. However, it vastly decreases upon increasing temperature and decreasing pH [1; 13]. Synthetic polyP molecules are linear, cyclic, or branched and have a chain length from three up to several thousand orthophosphate units [14]. Branched polyP, so-called ultraphosphates, have not been found in living organisms [15]. Their stability depends on pH and the presence or absence of divalent metal cations [16; 17]. It is currently unclear whether they are not formed in biological systems or are not found due to their short half-life in aqueous systems, as the branching sites are rapidly hydrolys [17]. Cyclic polyPs (i.e., metaphosphates) have been synthesised from linear polyP upon solvent removal, and they are comparably stable in aqueous solutions. The linear polyP backbone has a high degree of conformational flexibility with staggered and eclipsed conformations dependent on whether monovalent (e.g., NH\({}_{4}\)\({}^{+}\), Na\({}^{+}\), or K\({}^{+}\)), divalent (e.g., Mg\({}^{2+}\), Zn\({}^{2+}\), Ca\({}^{2+}\)), or even trivalent (e.g., Gd\({}^{3+}\)) cations are complexed to the phosphate backbone [18; 19]. Molecular modelling and experimental data revealed that binding Ca\({}^{2+}\) ions to polyP molecules leads to aggregate/microparticle formation ex vivo [20]. The length of a polyP molecule in its linear conformation can be estimated from the size of a single phosphate monomer (2.7 A), indicating that long-chain polyP polymers (around 1000 P\({}_{\text{i}}\) units) of bacterial and mammalian cell origin is up to \(\sim\)300 nm in length. However, the natural polymer is complexed to divalent cations and proteins that compact the polyP within subcellular compartments such as acidocalcisomes or platelet-dense granules. The phosphates in the polyP backbone are strongly acidic (pK\({}_{\text{a}}\)\(\sim\)0-3) molecules and are fully deprotonated at physiological pH [21; 22]. The negatively charged oxygen atoms from two adjacent phosphate groups complex divalent cations. Thus, polyP provides a scaffold for the local enrichment of Zn\({}^{2+}\) in platelet-dense granules and Ca\({}^{2+}\) in various intracellular compartments [23]. The complexed cations, at least in part, neutralise the polyanionic polymer backbone [24; 25; 26] (Figure 1). PolyP in living cells are linear polymers characterised by their mean chain length, distribution of chain length (dispersity), and binding partners, including cations and proteins. PolyP is ubiquitously found in higher organisms, including all mammalian species [28].