Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin

The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent i nhibitor of protein synthesis. This paper describes the use of a rabbit ret iculocyte 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 variab le slope (R-2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confid ence 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 o f toxin samples within the range 0.5 - 3.0 muM (200 - 1200 mug/L) CYN. To a ssess the usefulness of this assay, a range of toxic and nontoxic Cylindros permopsis raciborskii extracts, including both laboratory strains and envir onmental samples, were assayed by protein synthesis inhibition. These CYN q uantifications were then compared to quantifications obtained by high perfo rmance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLC MS-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) q uantifications. We conclude that this is an accurate and rapid assay for th e measurement of cylindrospermopsin in cyanobacterial extracts. (C) 2001 by John Wiley & Sons, Inc.