'iff. ..^i",- ■ 



22 

 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 

 using an Amicon stirred cell fitted with YM 10 filter. Concentrated PPO 

 was then dialyzed at 4°C overnight against 3 changes of 2L HjO. 



For grass prawn, the precipitate was resuspended in 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 was eluted with a stepwise gradient of elution buffer [100% 

 extraction buffer (9 mL), 50% extraction buffer in water (24 mL), and 10% 

 extraction buffer in water (24 mL)], 50% ethylene glycol (12 mL), and then 

 distilled water (150 mL) at a flow of 0.2 mL/min. Four-mL fractions were 

 collected and fractions exhibiting PPO activity were pooled and 

 concentrated via ultrafiltration utilizing an Amicon stirred cell fitted 

 with an XM 50 filter (Amicon). 



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