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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 (ε), 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 (λi = 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. Keywords: 3,4-Dihydroxyphenylpropionic acid; diphenols; enzyme kinetics; p-hydroxyphenylpropionic acid; MBTH; monophenols; mushroom; polyphenol oxidase; spectrophotometry; tyrosinase

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Abstract:The ability of the grapevine hypodermal peroxidase isoenzyme B, to oxidise the flavonols, quercetin and myricetin, and the flavonol glycosides quercetin-3-arabinoside, quercetin-3-rhamnoside (quercitrin) and myricetin-3-rhamnoside (myricitrin), has been studied. The results showed that, whilst the aglycones were good substrates of this peroxidase isoenzyme, their corresponding glycosides were poorly oxidised under the assay conditions. The oxidation of quercetin and myricetin catalysed by the hypodermal peroxidase isoenzyme B, was strictly dependent on hydrogen peroxide (H,O,). The kinetic characteristics of the flavonol oxidation were also studied. Whilst the apparent K, values for quercetin and myricetin were roughly equal (38 and 54 ,UM, respectively), the apparent K, values for H,O, in each reaction were appreciably different (168 and 8 ,UM, respectively). Besides, the optimal pH for quercetin oxidation was 4.0 whereas for myricetin it was 6.G7.0. The results are discussed in view of the possible involvement of the vacuolar hypodermal B, isoperoxidase in the turnover of flavonol and flavonol glycosides in grapes.

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