94 



against 3 changes of 2L of elution buffer. Column chromatography was 

 carried out at 4°C. 



Extraction and Purification of Apple PPO 



The modified method of Stelzig et al . (1972) was followed to purify 

 apple PPO. Apple peel (150 g) was mixed with liquid nitrogen and ground 

 into fine powder using a Waring blender. Powder (4 g) was homogenized at 

 4°C in 200 mL of 0.1 M sodium phosphate buffer (pH 6.0) containing 0.3 M 

 sucrose, 0.2% cysteine hydrochloride and 1 mL of 2 x 10"^ M 2-mercapto- 

 benzothiazole (MBT) in 95% ethanol , followed by centrifugation at 20,000g 

 (4°C) for 20 min. The pellet was washed twice with 50 mL 0.1 M phosphate 

 buffer (pH 6.0) containing 0.2% cysteine-HCl and resuspended in this 

 buffer. This suspension was made to 2% with Triton X-100 (Sigma), and 

 then incubated at 25°C for 15 min. Following centrifugation at 40,000g 

 (4°C) for 30 min, supernatant was collected and dialyzed at 4°C overnight 

 against 3 changes of 4L distilled H^O. The dialysate was extracted with 

 cold (-20°C) /j-butanol, the aqueous phase was collected and further 

 dialyzed overnight at 4°C against H2O. 



Hvdroxvl apatite Chromatography 



A hydroxy 1 apatite gel (Bio-Gel HT hydroxyl apatite, Bio-Rad) 

 suspension (200 mL) was poured into a K 40/26 column. It was then washed 

 with 0.001 M sodium phosphate buffer (pH 7.6). Crude apple PPO was loaded 

 onto the column, and 250 mL of 0.005 - 0.3 M (linear gradient) sodium 

 phosphate buffer (pH 7.6) containing 5% ammonium sulfate was used to 

 desorb the enzyme at a flow rate of 24 mL/hr. Four-mL fractions were 



