Fluorometric assay for the determination of glutathione reductase activity

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The determination of free sulfhydryl groups is important in many aspects of biotechnology, such as measuring the coupling efficiency of thiol-reactive probes, assaying cysteine-containing haptens, and assaying reductase/thiol transferase activity, as well as in various aspects of the food industry. This is generally achieved colorimetrically using Ellman's reagent, although the assay is relatively insensitive and Ellman's reagent is unstable. In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop a sensitive assay for glutathione reductase activity. The assay exploits the specific increase in fluorescence intensity that occurs at 520 nm when a probe containing two molecules of fluorescein linked via a disulfide group is cleaved by glutathione. The assay is 2 orders of magnitude more sensitive than the commonly used colorimetric glutathione reductase assay and has a greater dynamic range.

LanguageEnglish
Pages8769-8773
Number of pages5
JournalAnalytical Chemistry
Volume79
Issue number22
DOIs
Publication statusPublished - 15 Nov 2007

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Glutathione Reductase
Assays
Dithionitrobenzoic Acid
Sulfhydryl Compounds
Haptens
Biotechnology
Transferases
Fluorescein
Disulfides
Glutathione
Cysteine
Oxidoreductases
Fluorescence
Molecules
Industry

Cite this

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title = "Fluorometric assay for the determination of glutathione reductase activity",
abstract = "The determination of free sulfhydryl groups is important in many aspects of biotechnology, such as measuring the coupling efficiency of thiol-reactive probes, assaying cysteine-containing haptens, and assaying reductase/thiol transferase activity, as well as in various aspects of the food industry. This is generally achieved colorimetrically using Ellman's reagent, although the assay is relatively insensitive and Ellman's reagent is unstable. In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop a sensitive assay for glutathione reductase activity. The assay exploits the specific increase in fluorescence intensity that occurs at 520 nm when a probe containing two molecules of fluorescein linked via a disulfide group is cleaved by glutathione. The assay is 2 orders of magnitude more sensitive than the commonly used colorimetric glutathione reductase assay and has a greater dynamic range.",
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Fluorometric assay for the determination of glutathione reductase activity. / Piggott, Andrew M.; Karuso, Peter.

In: Analytical Chemistry, Vol. 79, No. 22, 15.11.2007, p. 8769-8773.

Research output: Contribution to journalArticleResearchpeer-review

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