title: Discrimination of extended-spectrum beta-lactamases by a novel nitrocefin competition assay.
abstract: We describe a nitrocefin competition assay for determining inhibition profiles as a useful adjunct to existing biochemical methods for the discrimination of beta-lactamases. The hydrolysis rate of nitrocefin was measured with a plate photometer as the change in A480 over 45 min in the presence of 17 inhibitors. Fourteen well-established beta-lactamases and 13 extended-spectrum beta-lactamases were tested. Correlations with data from isoelectric focusing and amino acid sequencing suggested that the inhibition profile reflects alterations in the active-site configuration of beta-lactamases. The method was especially useful in measuring the relative affinities of beta-lactamases against poorly hydrolyzed substrates and in screening large numbers of isolates for the detection of new beta-lactamase types.
authors.affiliation: Division of Infectious Diseases and Clinical Microbiology, Miriam Hospital, Providence, Rhode Island.
authors.forename: G A
authors.initials: GA
authors.lastname: Papanicolaou
authors.forename: A A
authors.initials: AA
authors.lastname: Medeiros
mesh_terms: D001667:Binding, Competitive; D002511:Cephalosporins; D002876:Chromosomes, Bacterial; D006868:Hydrolysis; D007525:Isoelectric Focusing; D001618:beta-Lactamases / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D002511:Cephalosporins; D001618:beta-Lactamases; C021720:nitrocefin