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University of Illinois – Urbana-Champaign

1. Hebbard, Carleigh Fredda Frances. Mechanisms of polyphosphate degradation.

Degree: PhD, Biochemistry, 2016, University of Illinois – Urbana-Champaign

The goal of my research has been to investigate the mechanisms by which inorganic polyphosphate (polyP) is degraded in vivo. PolyP is a linear chain of phosphate moieties linked through high-energy phosphoanhydride bonds and can range in size from three-phosphates-long to thousands. Naturally, these molecular chains are stored in cells within organelles called acidocalcisomes—metal-filled acidic cellular compartments. Human platelets, for example, store polyP of 60-100mer in dense granules (a type of acidocalcisome); upon activation, platelets release the procoagulant molecule into blood. Once in blood, polyP presumably decays. Previous studies indicated that polyP incubated in human serum has a half-life of about 90 minutes. Our hypothesis, therefore, was that native serum endo- and exopolyphosphatases degraded polyP. To study the degradation of polyP in vivo and identify putative polyphosphatases, we needed to develop a high-throughput method for measuring polyphosphatase activity. The study of polyP long has been hampered by the paucity of high-throughput methods for detecting and quantifying rates of polyphosphate degradation. Adapting carboxylic acid chemistry to esterify polyP's terminal phosphates with alcohols, we created chromogenic and fluorogenic polyphosphatase substrates that allow one to measure real-time activity of either endo- or exopolyphosphatases, depending upon assay configuration. We confirmed the products' identities through 1D and 2D 31P, 1H, and 13C NMR analyses. In proof-of-principle experiments we showed that the substrates were useful for spectrophotometrically monitoring the activities of commercially-available polyphosphatases in real time. Utilizing these substrates, we identified a new function for the clinically significant enzyme, Nudt2. The chemistry and substrates developed would be not only applicable for synthetic and clinical applications, but also for identifying putative human sera polyphosphatases. In attempting to identify the putative sera polyphosphatases, we found that the majority of polyphosphate degradation in serum was metal-mediated, rather than enzyme-mediated. Using a 96-well plate format assay, we tested a variety of conditions. PolyP degradation in serum had the following characteristics: resistance to canonical phosphatase inhibitors, resistance to heat, a calcium dependency, and a pH dependency. Our results show that at physiological pH (7.4) and above, calcium concentrations near and above those in serum (x ≥ 1.25 mM) can hydrolyze polyphosphate chains. Most unicellular organisms and many human cells (e.g. mast cells, platelets) store polyP with divalent metals in acidocalcisomes, and the dissolution of acidocalcisome polyP in response to alkaline stress occurs in various organisms and in situ marine mineral sedimentation. Until now, the reigning hypothesis has been that this polyphosphate degradation is enzyme-catalyzed. Our findings raise a question, though, as to whether or not calcium ions alone may be sufficient to facilitate polyP… Advisors/Committee Members: Morrissey, James H (advisor), Morrissey, James H (Committee Chair), Grosman, Claudio (committee member), Kranz, David M (committee member), Olsen, Gary J (committee member).

Subjects/Keywords: polyphosphate; polyP; Nudt2; Nudt3; pNP; p-nitrophenol; 4-nitrophenol; Nudix; phosphoanhydride; phosphoester; chromogenic substrate; fluorogenic substrate; phosphatase substrate; spectrophotometry; DAPI; 4-methylumbeliferone; dinucleotide polyphosphate; diadenosine polyphosphate; phosphate; phosphatase; phosphoanhydrase; phosphodiesterase; alkaline phosphatase; acid phosphatase; endopolyphosphatase; exopolyphosphatase; calcium; acidocalcisome; volutin granule; azurophilic granule; chitin; Chs2; ScChs2; cell wall; polysaccharide; oligosaccharide; calcium phosphate sink; ocean phosphate; serum; GlcNAc; UDP-GlcNAc; platelet; clotting; blood; coagulation; coagulation cascade; processive; polymer; TLC; carbohydrate; primer; TCA-insoluble; YO1531; inositol; inositol phosphate; ApnA; Ap3A; Ap4A; Ap6A; NpnA; diadenosine tetraphosphate; diadenosine pentaphosphate; diadenosine hexaphosphate; phytic acid; metaphosphate; phosphate melt; inositol hexaphosphate; 5-phosphoribosyl 1-pyrophosphate; PNPP; p-nitrophenol phosphate; DIPP; diphosphoinositol polyphosphate phosphohydrolase; Ddp1p; YOR163w; Saccharomyces cerevisiae; GlcN; GalNAc; ManNAc; Glc; GlcNAc2; GlcNAc3; N-propanoylglucosamine; GlcNPr; N-butanoylglucosamine; GlcNBu; N-glycolylglucosamine; GlcNGc

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APA (6th Edition):

Hebbard, C. F. F. (2016). Mechanisms of polyphosphate degradation. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from

Chicago Manual of Style (16th Edition):

Hebbard, Carleigh Fredda Frances. “Mechanisms of polyphosphate degradation.” 2016. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed November 14, 2019.

MLA Handbook (7th Edition):

Hebbard, Carleigh Fredda Frances. “Mechanisms of polyphosphate degradation.” 2016. Web. 14 Nov 2019.


Hebbard CFF. Mechanisms of polyphosphate degradation. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2016. [cited 2019 Nov 14]. Available from:

Council of Science Editors:

Hebbard CFF. Mechanisms of polyphosphate degradation. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2016. Available from: