Spectrophotometric method for the determination of polyphenol oxidase activity by coupling of 4-tert-butyl-o-benzoquinone and 4-amino-N,N- diethylaniline

We describe a spectrophotometric analytical method for the detection of polyphenol oxidase activity in an aqueous solution. The assay is based on the coupling reaction between 4-tert-butyl-o-benzoquinone, generated during the enzyme-catalyzed reaction acting on 4-tert-butylcatechol, and the aromatic amine, 4-amino-N,N-diethylaniline to yield a blue adduct (λ(max) 625 nm). This blue adduct exhibited spectral features different from the parent phenol and from the o-quinone. Polyphenol oxidase activities extracted from potatoes, and sunflower seedlings were assayed. The proposed method presents several advantages over the spectrophotometric measurement of 4-tert-butyl-o- benzoquinone. The duration of the linear period was increased, allowing a better determination of its value. The molar extinction coefficient of the blue adduct was higher than that of the 4-tertbutyl-o-benzoquinone; therefore the limitation imposed by the comparatively low e for the 4-tert-butyl-o- benzoquinone is overcome. This is of great importance when considering the inertness of the 4-tert-butyl-o-benzoquinone towards some common coupling agents such as 3-methyl-benzothiazolin-2-one hydrazone.