Aminoglycoside antibiotic measurement by bioluminescence, with use of plasmid-coded enzymes.
We describe a bioluminescent assay for gentamicin in serum that is applicable to the measurement of other aminoglycosides as well. The assay is based on the measurement of residual ATP with the luciferase reaction after incubation of the antibiotic with a plasmid-coded enzyme. Two aminoglycoside-inactivating enzymes were used: an adenylytransferase and an acetyltransferase coupled to S-acetyl coenzymeA synthetase. We investigated the latter system further because of the good stability of the acetyltransferase, its recovery in high yield from bacteria, and its more favorable ATP/gentamicin mass ratio. Serum ATPases were inactivated at 60 degrees C for 20 min. The operating range of the assay was 0-15 mg of gentamicin per liter. The precision (CV) was 10.1% at a concentration of 2 mg/L and 1.1% at 10 mg/L. The method correlated well with a radio-enzymatic assay for mock unknown sera (r = 0.981). The results were available within 2 h.
D000106:Acetate-CoA Ligase / Q000378:metabolism; D000123:Acetyltransferases / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000097:blood; D004926:Escherichia coli / Q000201:enzymology; D005839:Gentamicins / Q000097:blood*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D008722:Methods; D009713:Nucleotidyltransferases / Q000378:metabolism; D010957:Plasmids
D005839:Gentamicins; D008156:Luciferases; D000123:Acetyltransferases; D009713:Nucleotidyltransferases; D000251:Adenosine Triphosphatases; D000106:Acetate-CoA Ligase