Cell-free Protein Synthesis Inhibition Assay for the Cyanobacterial Toxin Cylindrospermopsin

S.M. Froscio, A.R. Humpage, Philip Burcham, I.R. Falconer

    Research output: Contribution to journalArticle

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    Abstract

    The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R-2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (CI) = 111 - 130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5 - 3.0 muM (200 - 1200 mug/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r(2) = 0.99) and HPLC (r(2) = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts. (C) 2001 by John Wiley & Sons, Inc.
    Original languageEnglish
    Pages (from-to)408-412
    JournalEnvironmental Toxicology
    Volume16
    DOIs
    Publication statusPublished - 2001

    Fingerprint

    toxin
    Assays
    assay
    protein
    High performance liquid chromatography
    Proteins
    liquid chromatography
    High Pressure Liquid Chromatography
    Cylindrospermopsis
    Protein Synthesis Inhibitors
    Poisons
    Reticulocytes
    Tandem Mass Spectrometry
    cylindrospermopsin
    cyanobacterial toxin
    Inhibitory Concentration 50
    Mass spectrometry
    Limit of Detection
    inhibitor
    mass spectrometry

    Cite this

    @article{5633bb571ea849599a635f50ff112490,
    title = "Cell-free Protein Synthesis Inhibition Assay for the Cyanobacterial Toxin Cylindrospermopsin",
    abstract = "The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R-2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95{\%} confidence limits (CI) = 111 - 130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5 - 3.0 muM (200 - 1200 mug/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r(2) = 0.99) and HPLC (r(2) = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts. (C) 2001 by John Wiley & Sons, Inc.",
    author = "S.M. Froscio and A.R. Humpage and Philip Burcham and I.R. Falconer",
    year = "2001",
    doi = "10.1002/tox.1050",
    language = "English",
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    Cell-free Protein Synthesis Inhibition Assay for the Cyanobacterial Toxin Cylindrospermopsin. / Froscio, S.M.; Humpage, A.R.; Burcham, Philip; Falconer, I.R.

    In: Environmental Toxicology, Vol. 16, 2001, p. 408-412.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Cell-free Protein Synthesis Inhibition Assay for the Cyanobacterial Toxin Cylindrospermopsin

    AU - Froscio, S.M.

    AU - Humpage, A.R.

    AU - Burcham, Philip

    AU - Falconer, I.R.

    PY - 2001

    Y1 - 2001

    N2 - The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R-2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (CI) = 111 - 130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5 - 3.0 muM (200 - 1200 mug/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r(2) = 0.99) and HPLC (r(2) = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts. (C) 2001 by John Wiley & Sons, Inc.

    AB - The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R-2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (CI) = 111 - 130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5 - 3.0 muM (200 - 1200 mug/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r(2) = 0.99) and HPLC (r(2) = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts. (C) 2001 by John Wiley & Sons, Inc.

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