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 pol yphenol 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 (lambda(max) 625 nm). This blue adduct exhibited spectral features different from the parent phenol and from the o-quinone. Polyphenol oxidase activities extracted fro m potatoes, and sunflower seedlings were assayed. The proposed method prese nts several advantages over the spectrophotometric measurement of 4-tert-bu tyl-o-benzoquinone. The duration of the linear period was increased, allowi ng a better determination of its value. The molar extinction coefficient of the blue adduct was higher than that of the 4-tert-butyl-o-benzoquinone; t herefore the limitation imposed by the comparatively low epsilon for the 4- tert-butyl-o-benzoquinone is overcome. This is of great importance when con sidering the inertness of the 4-lert-butyl-o-benzocluinone towards some com mon coupling agents such as 3-methyl-benzothiazolin-2-one hydrazone.