Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput Detection of Enzymes That Hydrolyze Inorganic Polyphosphate

Abstract

Inorganic polyphosphates, linear polymers of orthophosphate, occur naturally throughout biology and have many industrial applications. Their biodegradable nature makes them attractive for a multitude of uses, and it would be important to understand how polyphosphates are turned over enzymatically. Studies of inorganic polyphosphatases are, however, hampered by the lack of high-throughput methods for detecting and quantifying rates of polyphosphate degradation. We now report chromogenic and fluorogenic polyphosphate substrates that permit spectrophotometric monitoring of polyphosphate hydrolysis and allow for high-throughput analyses of both endopolyphosphatase and exopolyphosphatase activities, depending on assay configuration. These substrates contain 4-nitrophenol or 4-methylumbelliferone moieties that are covalently attached to the terminal phosphates of polyphosphate via phosphoester linkages formed during reactions mediated by EDAC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide). This report identifies Nudt2 as an inorganic polyphosphatase and also adds to the known coupling chemistry for polyphosphates, permitting facile covalent linkage of alcohols with the terminal phosphates of inorganic polyphosphate.

Dissolved Organic Phosphorus Utilization by Phytoplankton Reveals Preferential Degradation of Polyphosphates Over Phosphomonoesters

“…By quantifying the inert, signal-bearing probe that is released during P hydrolysis, these substrates can be used to track P-hydrolysis rates independently of P dynamics, which is especially important at high cell densities. Synthesis of labeled inorganic poly-P has been described (Choi et al, 2010;Hebbard et al, 2014), but these substrates were unreactive in positive controls tested in this study (data not shown).…”

Delivery of Inorganic Polyphosphate into Cells Using Amphipathic Oligocarbonate Transporters

“…To prepare diamine-linked polyP samples 1b – 3b , polyP 12, 22, and 45 were coupled to 2,2-(ethylenedioxy)bis(ethylamine) 4 by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-mediated (EDAC-mediated) phosphoramide coupling using a procedure modified from Hebbard et al 38 The 31 P NMR spectra of the resulting bis-phosphoramide end-capped polyP conjugates showed complete conversion to polyP-diamines 1b – 3b , as determined by the appearance of the characteristic P–N resonance at −2.0 ppm. The polyP-diamines were purified by precipitation into ethanol, rather than acetone 39 to avoid imine formation, which was identified as a side reaction with acetone.…”

NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes

“…Other in vitro substrates for NUDT2 whose levels might be affected by its loss include Ap 5 A, Ap 6 A, other homodinucleoside polyphosphates with four or more phosphoryl groups, e.g. diguanosine tetraphosphate (Gp 4 G) [ 4 , 5 ], inorganic polyphosphate [ 151 ] and phosphoribosyl pyrophosphate [ 152 ], but there is no evidence to suggest that these are significant substrates in vivo [ 4 ]. Ap 5 A and Ap 6 A appear to be confined to secretory granules in certain specialized cells while no specific mechanisms are known for the synthesis of Gp 4 G and other homodinucleoside polyphosphates in mammalian cells [ 4 , 5 ].…”