Cargando…
Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput Detection of Enzymes That Hydrolyze Inorganic Polyphosphate
[Image: see text] 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 ove...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2014
|
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4130250/ https://www.ncbi.nlm.nih.gov/pubmed/25000340 http://dx.doi.org/10.1021/bm500872g |
_version_ | 1782330306560262144 |
---|---|
author | Hebbard, Carleigh F. F. Wang, Yan Baker, Catherine J. Morrissey, James H. |
author_facet | Hebbard, Carleigh F. F. Wang, Yan Baker, Catherine J. Morrissey, James H. |
author_sort | Hebbard, Carleigh F. F. |
collection | PubMed |
description | [Image: see text] 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. |
format | Online Article Text |
id | pubmed-4130250 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-41302502015-07-07 Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput Detection of Enzymes That Hydrolyze Inorganic Polyphosphate Hebbard, Carleigh F. F. Wang, Yan Baker, Catherine J. Morrissey, James H. Biomacromolecules [Image: see text] 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. American Chemical Society 2014-07-07 2014-08-11 /pmc/articles/PMC4130250/ /pubmed/25000340 http://dx.doi.org/10.1021/bm500872g Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) |
spellingShingle | Hebbard, Carleigh F. F. Wang, Yan Baker, Catherine J. Morrissey, James H. Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput Detection of Enzymes That Hydrolyze Inorganic Polyphosphate |
title | Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput
Detection of Enzymes That Hydrolyze Inorganic Polyphosphate |
title_full | Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput
Detection of Enzymes That Hydrolyze Inorganic Polyphosphate |
title_fullStr | Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput
Detection of Enzymes That Hydrolyze Inorganic Polyphosphate |
title_full_unstemmed | Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput
Detection of Enzymes That Hydrolyze Inorganic Polyphosphate |
title_short | Synthesis and Evaluation of Chromogenic and Fluorogenic Substrates for High-Throughput
Detection of Enzymes That Hydrolyze Inorganic Polyphosphate |
title_sort | synthesis and evaluation of chromogenic and fluorogenic substrates for high-throughput
detection of enzymes that hydrolyze inorganic polyphosphate |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4130250/ https://www.ncbi.nlm.nih.gov/pubmed/25000340 http://dx.doi.org/10.1021/bm500872g |
work_keys_str_mv | AT hebbardcarleighff synthesisandevaluationofchromogenicandfluorogenicsubstratesforhighthroughputdetectionofenzymesthathydrolyzeinorganicpolyphosphate AT wangyan synthesisandevaluationofchromogenicandfluorogenicsubstratesforhighthroughputdetectionofenzymesthathydrolyzeinorganicpolyphosphate AT bakercatherinej synthesisandevaluationofchromogenicandfluorogenicsubstratesforhighthroughputdetectionofenzymesthathydrolyzeinorganicpolyphosphate AT morrisseyjamesh synthesisandevaluationofchromogenicandfluorogenicsubstratesforhighthroughputdetectionofenzymesthathydrolyzeinorganicpolyphosphate |