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collected and protein profile of fractions were recorded at 280 nm; 

 fractions showing PPO activity were pooled and dialyzed overnight (4°C) 

 against 2L H,0. Dialysate was further concentrated using an Amicon stirred 

 cell fitted with a YM 10 filter. 



Extraction and Purif ication nf Grass Prawn PPn 



The method of Rolle et al . (1991) was followed. Heads of grass 

 prawn were frozen in liquid N^ and then ground to a fine powder in liquid 

 nitrogen using a Waring blender. Powder was suspended in 3 volumes (w/v) 

 of 0.05 M sodium phosphate buffer (pH 7.2) containing 1 M sodium chloride 

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

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

 fractionated with ammonium sulfate. Ammonium sulfate precipitating 

 between - 40% saturation was collected by centrifugation at 23,500g at 

 4°C for 30 min. Precipitate was resuspended in 50 mL extraction buffer 

 containing 40% ammonium sulfate. After homogenization using a Dounce 

 manual tissue grinder, the sample was centrifuged at 23,500g (4°C) for 20 

 min. The precipitate was homogenized in extraction buffer and centrifuged 

 as previously described. The resulting precipitate was homogenized in < 

 extraction buffer, then subjected to high performance hydrophobic 

 interaction chromatography at 4°C using a preparative Phenyl Sepharose 

 CL-4B (Sigma) column (K 16/40) attached to a Dionex gradient pump (Dionex 

 Corp., Sunnyvale, CA). The column was pre-equil ibrated with extraction 

 buffer. 



PPO in the column was eluted with a stepwise gradient of elution 

 buffer [100% extraction buffer (9 mL), 50% extraction buffer in water (24 



