Capillary electrophoresis-based enzyme assays for v-lactamase enzymes

Generic in-capillary as well as offline CE-based enzyme assays were developed for serine-v-lactamases and metallo-v-lactamases. The hydrolysis of benzylpenicillin to benzylpenicilloic acid was analyzed using 100 mM sodium phosphate solution, pH 6.0, as a background electrolyte. In-capillary assays employed an uncoated as well as a polyethylene oxide-coated capillary, while the offline assays employing long end and short end injection were performed in an uncoated capillary. Using procaine hydrochloride or 4-hydroxybenzoic acid as internal standard, the respective assays were validated with regard to linearity, LOD and LOQ, repeatability, precision, and accuracy. The assays were applied to the determination of the Michaelis-Menten parameters Km and Vmax of Bacillus cereus penicillinase as well as New Delhi metallo-v-lactamase 1 and Verona integrin-encoded metallo-v-lactamase 2. Furthermore, the inhibition of the enzymes by irreversible and competitive inhibitors was evaluated. Comparable data were obtained with all assays. The use of a simple substrate ensured broad applicability to the various types of v-lactamases.