CYSTEINE AS AN INHIBITOR OF ENZYMATIC BROWNING .1. ISOLATION AND CHARACTERIZATION OF ADDITION-COMPOUNDS FORMED DURING OXIDATION OF PHENOLICS BY APPLE POLYPHENOL OXIDASE

The oxidation of different phenols [4-methylcatechol (MC), chlorogenic acid (CG), (-)-epicatechin (EC), and (+)-catechin (CA)], catalyzed by apple polyphenol oxidase (PPO), was investigated in the presence of an excess of cysteine. The occurrence of one cysteine addition compound with the two former phenols and two cysteine addition products with the latter phenols was demonstrated by HPLC. In all cases, the formation of addition compound(s) was proportional to the degradation of the phenol. After purification by gel filtration on Trisacryl GF05, the structure of each cysteine conjugate was determined by H-1 NMR spectroscopy. After additional H-1 homonuclear decoupling and 2D homonuclear COSY experiments, it was shown unequivocally that adduct with MC was 5-S-cysteinyl-3,4-dihydroxytoluene. With CG, the structure was 2-S-cysteinylchlorogenic acid. With catechins, the cysteine was attached on the B ring for the two conjugates. The 2'-position was involved in the first addition compound, whereas it was the 5'-position in the second one. Moreover, the study of UV spectra of the purified compounds allowed the conclusion that the two conjugates of each catechin were formed at the same rate in equivalent amounts.