Sensitive Assay for the Lactonase Activity of Serum Paraoxonase 1 (PON1) by Harnessing the Fluorescence Turn-On Characteristics of Bioorthogonally Synthesized and Geometrically Controlled Chemical Probes.

The lactonase activity of paraoxonase 1 (PON1) has a crucial antiatherogenic function, and also serves as an important biochemical marker in human blood because the aberrant lactonase activity of PON1 is a key indicator for a number of diverse human diseases. However, no sensitive fluorescence assays that detect PON1 lactonase activity are available. We report the synthesis of two fluorescence turn-on chemical probes and () able to quantify PON1 lactonase activity. The chemical probes were constructed utilizing a disulfide-containing bicyclononyne, derivatives of rhodamine B and carboxyfluorescein, and reactions including copper-free azide-alkyne cycloaddition. Fluorescence quenching in was characterized by spectroscopic studies and was mainly attributed to the effect of contact quenching. Kinetic analysis of confirmed the outstanding reactivity and specificity of with thiols in the presence of general base catalysts. The -based assay was employed to determine PON1 lactonase activity, with a linear range of 10.8-232.1 U L and detection limit (LOD) of 10.8 U L, to quantify serum PON1 activity in human sera, and to determine the of 20.9 μM for the 2-hydroxyquinoline inhibition of PON1 lactonase. We are employing to develop high-throughput assays for PON1 lactonase activity.