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.

journal_name

Biomacromolecules

journal_title

Biomacromolecules

authors

Hebbard CF,Wang Y,Baker CJ,Morrissey JH

doi

10.1021/bm500872g

subject

Has Abstract

pub_date

2014-08-11 00:00:00

pages

3190-6

issue

8

eissn

1525-7797

issn

1526-4602

journal_volume

15

pub_type

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