Substrate recycling scheme for tetrachloro-p-benzoquinone using bilirubin oxidase and NADH: Application for pentachlorophenol assay

A novel assay for tetrachloro-p-benzoquinone (TCBQ), the main oxidation pro duct of pentachlorophenol (PCP), was developed using bilirubin oxidase (BOX ) in the presence of excess NADH. TCBQ was easily and rapidly reduced by NA DH to 1,4-tetrachlorohydroquinone (TCHQ), which was then recycled back to T CBQ by the enzyme. BOX exhibited no reactivity toward NADH while its cataly tic activity for the oxidation of TCHQ was very high. Under an optimized co ndition (250 mu M 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 decre ase at 340 nm yielded a detection limit for TCBQ of 110 nM. Fluorescence de tection of the NADH using a lower enzyme concentration (0.1 U/mL) with exci tation and emission wavelengths of 345 and 450 nm, respectively, allowed fo r 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. Cou pling this oxidation reaction to the BOX/NADH assay attained PCP detection limits of 170 and 50 nM using absorbance and fluorescence measurements, res pectively. When tested on PCP-contaminated soil samples, the BOX assay comp ared very well with HPLC measurements.