A coupled enzymatic assay for salicylate and acetylsalicylate using salicylate hydroxylase and tyrosinase

Salicylate hydroxylase was used together with tyrosinase in a coupled enzymatic assay for determining salicylate and acetylsalicylate. In the presence of NADH and dioxygen, salicylate hydroxylase catalyzed the hydroxylation and decarboxylation of salicylate to catechol, which was then oxidized by tyrosinase to o-quinone. When NADH was used in excess, the resulting o-quinone product was recycled to its catechol form since o-quinone can oxidize NADH to produce NAD(+). Consequently, a cycle was established in which several NADH molecules were utilized by each catechol, which was easily followed by monitoring the rate of absorbance decrease at 340 nm. In comparison to its non-amplified counterpart, the recycling of catechol and o-quinone improved the detection limit of the spectrophotometric assay about ninety-fold. The method developed is a rapid, simple spectrophotometric assay with a linear response up to 1.4 mu M salicylate and detection limit of 6.5 nM. The recycling assay was applied to determine salicylate in plasma and urine samples and the results obtained agreed well with the established Trinder method.