m 



ml), and 10% extraction buffer in water (24 mL)], 50% ethylene glycol (12 

 mL), and then distilled water (150 mL) at a flow rate of 0.2 mL/min. 

 Four-mL fractions were collected and fractions exhibiting PPO activity 

 were pooled and concentrated via ultrafiltration utilizing a YM 10 filter. 



Extraction and Purification of Lobster PPO 



The modified procedures of Simpson et al . (1987) were followed. 

 Lobster cuticle was emerged in liquid nitrogen and ground into a fine 

 powder using a Waring blender. It was then extracted with 3 parts (w/v) 

 0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl and 0.2% Brij 

 35 at 4°C for 3 hr. Following centrifugation at 8,000g at 4°C for 30 min, 

 the supernatant was dialyzed at 4°C against 3 changes of 4L of 0.05 M 

 sodium phosphate buffer (pH 6.5). < 



PPO was purified using a nondenaturing preparative polyacryl amide 

 gel electrophoresis (PAGE). A one-mL aliquot of crude enzyme extract was 

 applied to each of eight gel tubes (12 cm length x 14 mm i.d.) containing 

 5% acrylamide/ 0.13% bisacrylamide gel, and ran at a constant current of 

 10 mA/tube. PPO was visualized using a specific enzyme-substrate staining 

 method (Constantidines and Bedford, 1967) using 10 mM DL-^-3,4- 

 dihydroxyphenyl alanine (DL-DOPA) in 0.05 M sodium phosphate buffer (pH 

 6.5). Gels containing PPO were sectioned and homogenized in 0.05 M 

 phosphate buffer (pH 6.5) utilizing a Dounce tissue grinder. Following 

 filtration through a Whatman No. 4 filter paper, the filtrate was 

 concentrated using an Amicon stirred cell fitted with 10 K filter. The 

 lobster PPO was further purified by subjecting the concentrated filtrate 

 to PAGE at 7.5% acrylamide/ 0.2% bisacrylamide gel. 



