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 th e 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 exces s, the resulting o-quinone product was recycled to its catechol form s ince o-quinone can oxidize NADH to produce NAD(+). Consequently, a cyc le was established in which several NADH molecules were utilized by ea ch catechol, which was easily followed by monitoring the rate of absor bance decrease at 340 nm. In comparison to its non-amplified counterpa rt, the recycling of catechol and o-quinone improved the detection lim it of the spectrophotometric assay about ninety-fold. The method devel oped is a rapid, simple spectrophotometric assay with a linear respons e up to 1.4 mu M salicylate and detection limit of 6.5 nM. The recycli ng assay was applied to determine salicylate in plasma and urine sampl es and the results obtained agreed well with the established Trinder m ethod.