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New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates
Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatograph...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371215/ https://www.ncbi.nlm.nih.gov/pubmed/30609080 http://dx.doi.org/10.1002/pro.3569 |
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author | Kawahara‐Nakagawa, Yuka Nishikawa, Koji Nakashima, Satoru Inoue, Shota Ohta, Takehiro Ogura, Takashi Shigeta, Yasuteru Fukutani, Katsuyuki Yagi, Tatsuhiko Higuchi, Yoshiki |
author_facet | Kawahara‐Nakagawa, Yuka Nishikawa, Koji Nakashima, Satoru Inoue, Shota Ohta, Takehiro Ogura, Takashi Shigeta, Yasuteru Fukutani, Katsuyuki Yagi, Tatsuhiko Higuchi, Yoshiki |
author_sort | Kawahara‐Nakagawa, Yuka |
collection | PubMed |
description | Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox‐dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non‐invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H(2) and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase‐mediated reactions simultaneously. Using the proposed system, we confirmed that H(2) (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D(2)/H(2)O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (k (out)) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates. |
format | Online Article Text |
id | pubmed-6371215 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-63712152019-02-26 New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates Kawahara‐Nakagawa, Yuka Nishikawa, Koji Nakashima, Satoru Inoue, Shota Ohta, Takehiro Ogura, Takashi Shigeta, Yasuteru Fukutani, Katsuyuki Yagi, Tatsuhiko Higuchi, Yoshiki Protein Sci Methods and Applications Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox‐dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non‐invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H(2) and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase‐mediated reactions simultaneously. Using the proposed system, we confirmed that H(2) (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D(2)/H(2)O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (k (out)) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates. John Wiley and Sons Inc. 2019-01-16 2019-03 /pmc/articles/PMC6371215/ /pubmed/30609080 http://dx.doi.org/10.1002/pro.3569 Text en © 2019 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Methods and Applications Kawahara‐Nakagawa, Yuka Nishikawa, Koji Nakashima, Satoru Inoue, Shota Ohta, Takehiro Ogura, Takashi Shigeta, Yasuteru Fukutani, Katsuyuki Yagi, Tatsuhiko Higuchi, Yoshiki New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates |
title | New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates |
title_full | New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates |
title_fullStr | New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates |
title_full_unstemmed | New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates |
title_short | New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates |
title_sort | new assay method based on raman spectroscopy for enzymes reacting with gaseous substrates |
topic | Methods and Applications |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371215/ https://www.ncbi.nlm.nih.gov/pubmed/30609080 http://dx.doi.org/10.1002/pro.3569 |
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