PPO Extract Preparation and Assay. Apple PPO was extracted
from an apple acetone powder following the method of Yoruk et al.
(14). Enzyme activity was determined spectrophotometrically using
catechol as the substrate. The 3 mL standard reaction mixture, prepared
in a cuvette, contained 2.35 mL of acetate buffer, 0.3 mL of SC solution
(or 0.3 mL acetate buffer for the control), 0.3 mL of catechol solution
in acetate buffer, and 0.05 mL of PPO extract. The reference cuvette
contained only the catechol solution and acetate buffer. The changes
in absorbance at 420 nm and 25 °C for 3 min were measured using a
Shimadzu PharmaSpec UV-1700 UV-vis spectrophotometer (Shimadzu
Scientific Instruments, Inc., Columbia, MD). The enzyme
activity was determined on the basis of the initial reaction rates.