54 



Purification of Mushroom, Potato, Lobster, and Shrimp PPO 



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 Labs.) and a 

 power supply (Model EPS 500/400, Pharmacia LKB Biotechnology Inc.). A 

 one-mL aliquot of crude enzyme extract (potato, lobster, or shrimp) was 

 applied to each of eight gel tubes (1.4 cm I.D. x 12 cm length) containing 

 5% acryl amide/ 0.13% bisacryl amide gel and subjected to a constant current 

 of 10 mA/tube in a buffer (pH 8.3) containing 5 mM Tris-HCl and 38 mM 

 glycine (Sigma Chemical CO., 1984). PPO was visualized using a specific 

 enzyme-substrate staining method (Constantinides and Bedford, 1967); 10 mM 

 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 

 0.05 M sodium phosphate buffer (pH 6.5) utilizing a Dounce manual tissue 

 grinder. The homogenates were filtered through Whatman No. 4 filter 

 paper, pooled, and concentrated using an Amicon stirred cell (Model 8050). 

 Mushroom PPO (0.25 mg/mL) in 0.05 M sodium phosphate buffer (pH 6.5) was 

 further purified according to the procedures previously described. 



Protein Quantitation and Enzyme Purity Determination 



Protein content of the various PPO preparations was 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). Mushroom, potato, and crustacean PPOs (20 /xg 



