Colorimetric assay for β-lactamase activity using cocktail of penicillin and 4-(2-pyridylazo)resorcinol (PAR)–2Hg2+ complex

Abstract A colorimetric assay method for measuring β-lactamase activity was developed using a cocktail of penicillin and 4-(2-pyridylazo)resorcinol (PAR)–2Hg 2+ complex. The penicilloic acid that hydrolyzed form of penicillin by β-lactamase chelates with mercury ion of PAR–2Hg 2+ complex effectively, conversion of PAR–2Hg 2+ complexes to free PAR induced the color change from red to yellow. Exact measurement of β-lactamase activity can be performed by the spectrophotometric method, and the facile monitoring of β-lactamase activity was possible in real time. Also, this colorimetric method was applied to determine the Michaelis constant ( K m ) of β-lactamase and the IC 50 of clavulanic acid. Given the facts that its simplicity and high assay speed, this method may be extended to high-throughput screening of β-lactamase inhibitors.