A firefly luciferase assay for subnanomolar concentrations of amphipathic substances

A sensitive assay is described for accurately quantitating subnanomolar aqueous concentrations of a wide variety of amphipathic and hydrophobic biological materials. This paper extends a luciferase-luciferin method previously used to measure aqueous concentrations of anesthetics to a variety of hormones, metabolites, and membrane active agents. The assay can cover analyte ranges from picomolar to micromolar. The sensitivity of the assay is shown to correlate with the hydrophobic nature of the analyte. The mechanism of the assay appears to result from competition of analyte with luciferin for a hydrophobic binding site on the luciferase molecule. This assay allows measurement of the partitioning of analytes into lipid bilayers from aqueous solution.