



supernatant was dialyzed at 4°C overnight against 3 changes of 4L distilled 



Crude PPO preparation was purified further using a nondenaturing 

 preparative polyacryl amide gel electrophoresis (PAGE) system. Equipment 

 utilized included a gel tube chamber (Model 175, Bio-Rad) and a power 

 supply (Model EPS 500/400, Pharmacia). A one-mL aliquot of crude enzyme 

 extract (lobster, shrimp, or potato) was applied to each of eight gel 

 tubes (1.4 cm I.D. x 12 cm length) containing 5% acrylamide/ 0.13% 

 bisacrylamide gel and run at a constant current of 10 mA/tube (Sigma 

 Chemical CO., 1984). PPO was visualized using a specific enzyme-substrate 

 staining method; 10 mM DL-^3,4-dihydroxyphenylalanine (DL-DOPA) in 0.05 

 M sodium phosphate buffer (pH 6.5) was used as substrate. After the 

 migration of the enzyme relative to the dye front (R,) was determined using 

 one of the eight gels, the remaining gels were sectioned at the determined 

 R,. PPO was eluted from the gel by homogenization in distilled water 

 utilizing a Bounce manual tissue grinder. The homogenates were filtered 

 through Whatman No. 4 filter paper, pooled, and concentrated using an ' 

 Amicon stirred cell (Model 8050) fitted with a YM 10 filter. Concentrated 

 PPO was dialyzed overnight (4°C) against 2 changes of 4L distilled water. 



Protein Quantitation and F p zyme Purity neterminatinn 



Protein contents of various PPO preparations were quantitated using 

 the Bio-Rad Protein Assay kit with bovine serum albumin as standard. 

 Enzyme purity was examined using a mini gel system (Mini -Protean II Dual 

 Slab Cell) (Bio-Rad, 1985b). PPO (20 ,g protein/well) was loaded and 

 electrophoresis was carried out at constant voltage (200 V) for 35 min. 



