20 

 Chemicals) which had been equilibrated with 1.0 mM potassium phosphate 

 buffer (pH 7.0). Unbound phenolic compounds and proteins were washed off 

 using 250 mL I mM phosphate buffer (pH 7.0) at 24 mL/hr for 3 hr. Elution 

 of PPO was performed using a linear gradient (0-1.0 M) of NaCl in 1.0 mM 

 potassium phosphate buffer at 24 mL/hr for 18 hr. Four-mL fractions were 

 collected, and protein was estimated by spectrophotometry at 280 nm. 

 Fractions showing PPO activity were pooled and concentrated using an 

 Amicon stirred cell fitted with an Amicon YM 10 filter. 



The partially purified enzyme preparation was loaded onto a Sephadex 

 G-lOO (Pharmacia) gel filtration column (Pharmacia K 26/40) 

 pre-equilibrated with 1.0 mM potassium phosphate buffer (pH 7.0). The 

 column was eluted at 4°C with 400 mL 1.0 mM potassium phosphate buffer (pH 

 7.0) at 24 mL/hr for 15 hr. Four-mL fractions were collected and protein 

 estimated as above; fractions showing PPO activity were pooled and 

 concentrated using an Amicon stirred cell. Concentrated samples were 

 dialyzed at 4''C overnight against 3 changes of 2L elution buffer. 



Extraction and Purification of Apple PPO . „ 



The method of Stelzig et al . (1972) with some modification was 

 followed. Crude apple PPO after partial purification and dialysis against . 

 HjO was loaded onto an HT hydroxyl apatite (Bio-Rad) column (K 25/40). The 

 enzyme was desorbed from the gel using 250 mL 0.005 - 0.3 M (linear 

 gradient) sodium phosphate buffer (pH 7.6) containing 5% ammonium sulfate 

 at 24 mL/hr for 18 hr. Four-mL fractions were collected and protein 

 estimated by absorbance at 280 nm; fractions showing PPO activity were 

 pooled and dialyzed overnight (4°C) against 2 changes of 4L HjO. The 



