97 



Extraction anH Pny ification nf White Shrim p PPn 



The combined methods of Simpson et al . (1987) and Rolle et al . 

 (1991) with slight modification were followed to purify white shrimp PPO. 

 Shrimp cephalothorax was frozen in liquid nitrogen and ground using a 

 Waring blender. Shrimp 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. 

 Precipitate collected 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. Elution of PPO was performed by a linear gradient (0 - 1.0 M) of 

 NaCl with 300 mL 0.05 M sodium phosphate buffer (pH 7.2). 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 YM 10 filter. 



PPO was loaded onto a Sephadex G-lOO gel column (K 26/40) pre- 

 equilibrated with 0.05 M sodium phosphate buffer (pH 7.2) and then eluted 

 with 300 mL 0.05 M sodium phosphate buffer (pH 7.2) at 0.15 mL/min. 

 Three-mL fractions were collected and protein was estimated by absorbance 

 at 280 nm; fractions showing PPO activity were pooled and concentrated 



