146 

 Purity of the preparations was determined by comparing gels stained with 

 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5) and then with a 

 Coomassie blue R-250 solution. 



Enzyme Activity accay 



PPO activities were measured by adding 60 /zL of the preparation to 

 840 ML 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5) and 

 monitored at 25°C for 5 min. Maximal initial velocity was determined as 

 AA475 nn/fni" and one unit of PPO activity was defined as an increase in 

 absorbance of 0.001/min at 25°C. Unless otherwise specified, experiments 

 were replicated three times. The enzyme activities of lobster, brown 

 shrimp, and potato PPO was determined as 3,750, 740, and 5,400 units/mg 

 protein, respectively. 



Effect o f CO ^ fl atm) on PPQ Activity 



Three batches of 20-mL lobster PPO (1,470 units/mg protein) were 

 loaded in 50-mL polymethylpentene tubes (Nalgene Co.) and were heated 

 respectively at 33°, 38°, and 43°C in a water-bath. Following the 

 equilibration of the PPO solution to the desired temperatures, liquid 

 carbon dioxide [Coleman grade (99.99% CO,), Liquid Air Co., Walnut, CA)] , 

 at a flow rate of 110 mL/min was bubbled through the PPO solution for 30 

 min. PPO (500 /iL) was removed every 5 min from the stock solution heated .:. 

 at 33° or 38°C. PPO (500 ^L) heated at 43°C was sampled every min during 

 the first 5 min and then 5 min thereafter. PPO samples were put in 1.5 ml 

 microcentrifuge tubes (Fisher) and immediately cooled by emerging the tube 

 in an ice bath. Following equilibration to ambient temperature, PPO 



