■ '* ';■ 



98 



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

 was then dialyzed at 4°C overnight against 3 changes of 2L H2O. 



Protein Quantitation and Enzyme Purity Determination '- 



Protein content of all 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). Plant and crustacean PPO (20 /xg protein/well) was 

 loaded and electrophoresis was carried out at constant voltage (200 V) for 

 35 min. The purity of enzyme preparations were determined by comparing 

 gels stained with 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5) 

 (Constantidines and Bedford, 1967) and then with a Commassie brilliant 

 blue R-250 solution. ' 



Enzyme Activity Assay 



Potato PPO activity was determined at 25°C for 5 min by mixing 2.9 

 mL 0.97 mM chlorogenic acid in 1 mM potassium phosphate buffer (pH 7.0) 

 with 0.1 mL enzyme. Maximal initial velocity for quinone formation was 

 monitored at 395 nm using a DU-7 spectrophotometer. One unit of PPO 

 activity was defined as an increase in absorbance of 0.001/min at 395 nm 

 and 25°C. Apple PPO activity was measured at 30°C for 5 min by mixing 0.2 

 mL PPO preparation with 1.8 mL of 0.05 M 4-methyl catechol in 0.1 M sodium 

 phosphate buffer (pH 6.0). Maximal initial velocity for quinone formation 

 was determined at 395 nm and one unit of PPO activity was defined as an 

 increase in absorbance of 0.001/min. 



