Improvement of a continuous spectrophotometric method for determining the monophenolase and diphenolase activities of mushroom polyphenol oxidase

A spectrophotometric method for determining the monophenolase and diphenolase activities of mushroom polyphenol oxidase (PPO) at pH 6.8 has been improved. The method is based on the coupling reaction between the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) and the quinone products of the oxidation of monophenols and o-diphenols in the presence of polyphenol oxidase. MBTH-quinone adduct is further oxidized by another molecule of o-quinone. Different o-diphenols were assayed: L-dopa, dopamine, catechol, 4-methylcatechol, 3,4-dihydroxyphenylacetic acid (DHPAA), and 3,4-dihydroxyphenylpropionic acid (DHPPA) (and their corresponding monophenols). The PHPPA (p-hydroxyphenylpropionic acid)/DHPPA pair was chosen as the best pair from those assayed thanks to its kinetic features, molar absorptivity (epsilon), and solubility. All the MBTH-o-quinone adducts from the above substrates evolved at pH 6.8. A reaction mechanism for explaining the evolution of the MBTH-o-quinone adduct of DHPPA has been proposed and kinetically studied for the first time. The wavelength where the MBTH-o-quinone adduct of DHPPA showed an isosbestic point (lambda(i) = 466 nm) was chosen for spectrophotometrically recording the action of PPO on the PHPPA/DHPPA pair. This method could be useful for determining microquantities of PPO in problem samples.