Modified Fluorimetric Assay for Estimating Ampicilloate Concentrations and its Use for Detecting β-Lactamase and Penicillin Acylase Activity in Bacteria

Abstract

Sodium ampicilloate concentrations were estimated fluorimetrically by heating solutions with ascorbic acid, EDTA and a modified Lowry A reagent which was prepared by including copper sulfate and potassium sodium tartrate in 0.5 mol dm ^{- 3} acetate buffer at pH 4. A concentration range of 0.5–50 µmol dm ^{- 3} was used for the estimations. The reaction was used to estimate β-lactamase activity on ampicillin but the substrate also showed some fluorescence and a calculation was required to determine the amount of ampicilloate formed when both substances were present in the one reaction mixture. The β-lactamase was inhibited by treatment with trichloroacetic acid so the procedure could be used to assay the enzyme activity after a fixed time. 6-Aminopenicillanic acid did not fluoresce on treatment with the modified reagent and organisms which contained penicillin acylase lowered the amount of ampicillin which could be converted to ampicilloate. When penicillin acylase and β-lactamase co-existed in the one organism, the respective activities were determined by use of the copper–ascorbate–EDTA fluorescence assay for ampicilloate coupled with a fluorescamine assay for 6-aminopenicillanic acid determinations. On prolonged incubation, some organisms containing penicillin acylases lowered the amount of ampicilloate which formed a fluorescent product. This effect was attributed to deacylation of ampicilloate by the penicillin acylases.