Electrophoretic assay for penicillinase: substrate specificity screening by parallel CE with an active pixel sensor.
We report application of a new UV imaging detector incorporating an active pixel sensor in an electrophoretic enzyme assay for penicillinase (beta-lactamase) with multiple substrates. The method based on electrophoretically mediated microanalysis was developed on a standard CE system with a single-point diode array detector and 200 nm UV wavelength, then transferred to a parallel capillary setup with the UV imaging detector for screening of penicillinase substrate specificity. One capillary is used for the assay and the other for reference, with an enzyme solution plug introduced into the first at the same time as a water plug into the second capillary. A mixture of antibiotics and markers is subsequently introduced as a sample plug to both capillaries, and driven through the enzyme (or water) plug by application of voltage. Most individual reactant and product peaks were separated and compounds amenable to beta-lactam hydrolysis could readily be identified and the extent of the reaction quantified within a single electrophoretic run.
Urban
Pawel L
PL
Department of Chemistry, University of York, Heslington, UK.
Goodall
David M
DM
Bergström
Edmund T
ET
Bruce
Neil C
NC
D000900:Anti-Bacterial Agents / Q000032:analysis*; D002021:Buffers; D019411:Clinical Laboratory Techniques; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis*; D005336:Fiber Optic Technology / Q000295:instrumentation; D017022:Flow Injection Analysis; D007202:Indicators and Reagents; D008832:Microchemistry; D009862:Online Systems; D010405:Penicillinase / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D002021:Buffers; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D010405:Penicillinase