

21 

 dialysate was further concentrated using an Amicon stirred cell fitted 

 with a YM 10 filter. '■ ; y 



Extraction and Purification of White Shrimp and Grass Prawn PPO 



The methods of Chen et al . (1991a) and Rolle et al . (1991) with 

 slight modification were followed to purify white shrimp and grass prawn 

 PPO, respectively. Shrimp cephalothorax powder was suspended in 3 volumes 

 (w/v) 0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl 

 (extraction buffer) and 0.2% (v/v) Brij 35, and stirred at 4''C for 3 hr. 

 Following centrifugation at 23,000g (4°C) for 30 min, the supernatant was 

 fractionated with ammonium sulfate between - 40% saturation; protein 

 precipitate was collected by centrifugation at 23,500g at 4°C for 30 min. 



For white shrimp, the precipitate was dissolved in 0.05 M phosphate 

 buffer (pH 7.2) and dialyzed at 4°C overnight against 3 changes of 4L of 

 0.05 M phosphate buffer (pH 7.2). The dialyzed PPO was loaded onto a 

 DEAE-cellulose (0.95 meq/g) column (K 26/40) pre-equilibrated with 0.05 M 

 phosphate buffer (pH 7.2). Sixty-mL of 0.05 M sodium phosphate buffer (pH 

 7.2) was used to desorb unbound phenolic compounds and proteins at 0.2 

 mL/min for 1.5 hr. Elution of PPO was performed using a 300 mL 0.05 M 

 sodium phosphate buffer (pH 7.2) containing a linear gradient (0 - 1.0 M) 

 of NaCl . Three-mL fractions were collected and the protein estimated by 

 absorbance at 280 nm. Fractions possessing PPO activity were pooled and 

 concentrated using an Amicon stirred cell fitted with a YM 10 filter. 



