High-Performance Liquid Chromatography Analysis of N-Acyl Homoserine Lactone Hydrolysis by Paraoxonases

Abstract

Mammalian paraoxonases (PONs) are a unique, highly conserved family of calcium-dependent esterases consisting of PON1, PON2, and PON3. The PONs can hydrolyze the lactone ring of a range of N-acyl-l-homoserine lactone (AHL) quorum sensing signaling molecules, rendering them inactive. This chapter describes a method that utilizes high-performance liquid chromatography analysis with UV detection for determining the rate of AHL hydrolysis in cell lysates, tissue homogenates, serum, and with purified proteins. Also described are the techniques used to prepare cell culture lysates and tissue homogenates for analysis and the use of class-specific enzyme inhibitors to determine the contribution of PONs to AHL hydrolysis in the samples.