Purification and Characterization of Latent Polyphenol Oxidase from Apricot (Prunus armeniaca L.)

Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO4 and low concentrations (<= 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 degrees C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (K-M = 2.0 mM) and chlorogenic acid (K-M = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 degrees C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37455.6 Da (Asp102 Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt 081103) by means of peptide mass fingerprinting.