The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays.
Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 microM (270 microg l(-1)). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml(-1) LDH and 0.8 U ml(-1) LOD in reactions containing 246 microM pyruvate and 7.5 microM NADPH. PCP detection limits were an EC(10) of 800 nM (213 microg l(-1)) and a minimum inhibition detectable (MID) limit of 650 nM (173 microg l(-1)). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 microM. PCP detection limits were obtained for an assay containing 5 U ml(-1) LDH, 0.8 U ml(-1) LOD and 0.1 U ml(-1) GDH with 246 microM pyruvate, 400 mM glucose and 2 microM NADPH. The EC(10) limit was 150 nM (39.9 microg l(-1)) and the MID was 100 nM (26.6 microg l(-1)). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.
Young
S J
SJ
Department of Environmental Science, Faculty of Applied Science, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, UK.
Hart
J P
JP
Dowman
A A
AA
Cowell
D C
DC
D015374:Biosensing Techniques / Q000379:methods*; D002482:Cellulose / Q000031:analogs & derivatives*; D004563:Electrochemistry; D004784:Environmental Monitoring / Q000379:methods; D004785:Environmental Pollutants / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000633:toxicity*; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors; D008567:Membranes, Artificial; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors; D008954:Models, Biological; D010416:Pentachlorophenol / Q000633:toxicity
D004785:Environmental Pollutants; D004791:Enzyme Inhibitors; D008567:Membranes, Artificial; C005062:acetylcellulose; D002482:Cellulose; D006861:Hydrogen Peroxide; D010416:Pentachlorophenol; D006899:Mixed Function Oxygenases; D007770:L-Lactate Dehydrogenase; C021497:lactate 2-monooxygenase