Improvement of a Continuous Spectrophotometric Method for Determining the Monophenolase and Diphenolase Activities of Mushroom Polyphenol Oxidase Juan Carlos Espı ´n, † Mercedes Morales, † Pedro Antonio Garcı ´a-Ruiz, ‡ Jose ´ Tudela, † and Francisco Garcı ´a-Ca ´ novas* ,† GENZ: Grupo de investigacio ´n Enzimologı ´a, Departamento de Bioquı ´mica y Biologı ´a Molecular-A, Facultad de Biologı ´a, Universidad de Murcia, Apto. 4021, E-30080 Espinardo, Murcia, Spain, and Departamento de Quı ´mica Orga ´ nica, Facultad de Quı ´mica, Universidad de Murcia, E-30080 Espinardo, Murcia, Spain 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 monophe- nols). The PHPPA (p-hydroxyphenylpropionic acid)/DHPPA pair was chosen as the best pair from those assayed thanks to its kinetic features, molar absorptivity (ǫ), 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 (λ 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. Keywords: 3,4-Dihydroxyphenylpropionic acid; diphenols; enzyme kinetics; p-hydroxyphenylpro- pionic acid; MBTH; monophenols; mushroom; polyphenol oxidase; spectrophotometry; tyrosinase INTRODUCTION Polyphenol oxidase (EC 1.14.18.1) (PPO) as present in plant tissues plays an important role in fruit and vegetable processing and during storage of the processed foods. Prevention of browning of foods, enzymatic or nonenzymatic, has long been the concern of food scien- tist (Matheis, 1987). PPO is a copper enzyme, which in the presence of oxygen catalyzes two different reac- tions: the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the oxidation of o-diphe- nols to o-quinones (diphenolase activity) which, in turn, are polymerized to brown, red, or black pigments (Mason, 1955; Prota, 1988). Moreover, the enzyme is of central importance in vertebrate pigmentation. It is directly responsible for the conversion of the amino acid tyrosine to one of several types of melanin pigments (Robb, 1984). Due to the poor specificity of PPO for the phenolic substrates, several assay methods have been devel- oped: oximetric methods (Mayer and Harel, 1979); spectrophotometric methods which can measure the appearance of the o-quinones (Waite, 1976; Cabanes et al., 1987) or aminechrome (Mason, 1948; Garcı ´a-Moreno et al., 1991; Rodrı ´guez-Lo ´pez et al., 1992a). Moreover, using 3,4-dihydroxymandelic acid as substrate, 3,4- dihydroxybenzaldheyde production has been monitored by HPLC (Czapla et al., 1991) and by spectrophotometry (Rodrı ´guez-Lo ´pez et al., 1991). Most of the above described methods are focused on the diphenolase activity of this enzyme. However, there are only a few works on monophenolase activity (Rod- rı ´guez-Lo ´pez et al., 1992a; Ros et al., 1994; Espı´n et al., 1995b) because the enzyme’s kinetic mechanism action on monophenols has been recently clarified (Rodrı ´guez- Lo ´pez et al., 1992a; Ros et al., 1994). This has led us to develop continuous methods to follow this activity. Recently we developed a continuous spectrophotomet- ric method to determine the monophenolase and diphe- nolase activities of mushroom PPO, using L-dopa and L-tyrosine as substrates and MBTH as coupled reagent. (Rodrı ´guez-Lo ´pez et al., 1994). In a further attempt to improve and apply this method to measure PPO activity from several fruits and vegetables, different substrates (including L-dopa, dopam- ine, catechol, 4-methylcatechol, and DHPPA) were used for apple PPO (Espı ´n et al., 1995a). In order to determine the molar absorptivities (ǫ) for their corre- sponding MBTH-o-quinone adducts, the substrates were depleted in the presence of MBTH and a high PPO concentration. When the experiments were carried out at acidic pH (∼5), the adducts were soluble and stable, whereas at the optimum pH for mushroom PPO, 6.8, the adducts evolved showing isosbestic points (Espı´n et al., 1995a). This latter fact may not be very significant when diphenolase activities were assayed at 60-200 s per recording but may be much more significant when the monophenolase activity is assayed, which usually requires more than 15 min of assay time. The aim of this paper is to determine the best substrate for mushroom PPO, to propose a reaction mechanism to explain the evolution of its corresponding * Author to whom correspondence should be ad- dressed. † Departamento de Bioquı ´mica y Biologı ´a. ‡ Departamento de Quı ´mica Orga ´ nica. 1084 J. Agric. Food Chem. 1997, 45, 1084-1090 S0021-8561(96)00428-1 CCC: $14.00 © 1997 American Chemical Society