New spectrophotometric assay for polyphenol oxidase activity

We describe a convenient and sensitive assay of polyphenol oxidase (PPO, EC 1.14.18.1) consisting of spectrophotometry at 300 nm based on the stoichiometric reaction of cysteine with o-quinones produced during the enzymatic oxidation of phenols. The adduct formed exhibited spectral properties different from those of the parent phenol. PPO activities extracted from apple, pear, and mushroom were assayed. The assay cannot be used with hydroxycinnamoyl derivatives since the cysteinyl adduct compounds exhibited spectral properties similar to those of their parent phenolic substrates. However, the cysteine-coupled method presents several advantages over the measurement of oxygen uptake by polarography or the direct estimation of o-quinone by spectrophotometry. The duration of the linear period was increased, allowing a better estimation of its value. The zone of proportionality between rates and enzyme quantities was enlarged. The difference in molar extinction coefficients between adduct and phenol at 300 nm ranged between 2000 and 2800 M-1.cm-1, i.e., two times higher than those of the corresponding o-quinones. Therefore, this assay improves the sensitivity of polyphenol oxidase detection over that of the direct spectrophotometric assay of quinone formation.