American Chemical Society, Environmental Science and Technology, 5(33), p. 796-800, 1999
DOI: 10.1021/es980978k
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A novel assay for tetrachloro-p-benzoquinone (TCBQ), the main oxidation product of pentachlorophenol (PCP), was developed using bilirubin oxidase (BOX) in the presence of excess NADH. TCBQ was easily and rapidly reduced by NADH to 1,4-tetrachlorohydroquinone (TCHQ), which was then recycled back to TCBQ by the enzyme. BOX exhibited no reactivity toward NADH while its catalytic activity for the oxidation of TCHQ was very high. Under an optimized condition (250 μM NADH, 0.3 U/mL BOX, and 25 mM sodium phosphate at pH 5.5), the rate of NADH consumption determined by measuring the absorbance decrease at 340 nm yielded a detection limit for TCBQ of 110 nM. Fluorescence detection of the NADH using a lower enzyme concentration (0.1 U/mL) with excitation and emission wavelengths of 345 and 450 nm, respectively, allowed for a TCBQ detection limit of 30 nM. PCP was oxidized to TCBQ with high yield using bis(trifluoroacetoxy)iodobenzene in 0.05 M trichloroacetic acid. Coupling this oxidation reaction to the BOX/NADH assay attained PCP detection limits of 170 and 50 nM using absorbance and fluorescence measurements, respectively. When tested on PCP-contaminated soil samples, the BOX assay compared very well with HPLC measurements.