The Detection of Pentachlorophenol by Its Inhibitory Effectiveness on Lactate Dehydrogenase of Rabbit Muscle and Bovine Heart

There is a requirement for on-line monitoring of pentachlorophenol (PCP) in water supplies. One method of PCP detection may be to use enzyme-inhibition biosensors. In the present study, assay conditions were optimised, comparing two isozymes of lactate dehydrogenase (LDH) with regard to inhibition by PCP. Rabbit muscle LDH displayed greater inhibition of enzyme activity when exposed to 70 muM PCP than bovine heart LDH. Differences in inhibition between the two isozymes could be attributed to the varying affinities for the reactants, for which heart LDH had lower Km values than the muscle isozyme. PCP exerted a mixed type of competitive-noncompetitive inhibition on each isozyme with respect to cofactor binding, in which Ki indicated the binding affinity of PCP for the free enzyme and Kies represented the affinity for the binary complex of enzyme and cofactor. PCP exhibited competitive inhibition with respect to substrate binding, in which Ki indicated the affinity of PCP for the substrate binding site of the binary complex. PCP displayed a preference for binding to the free enzyme. Modified assays incorporating a nonoptimum cofactor, betaNADPH, showed significantly increased inhibition compared to those with beta-NADH. The ratio of Ki: Km provided a measure to estimate the inhibitory effectiveness of PCP, for which a ratio of Ki < Km indicated a preference for PCP binding to the enzyme. Such a ratio was shown when beta-NADPH or alpha-ketobutyrate was incorporated into the enzyme assay. Inhibition was greatest when using rabbit muscle LDH and beta-NADPH and gave a PCP detection limit (EC10) of 270 mug L-1 (270 ppb). This value compares favourably with other detection systems and offers reduced assay variability.