

protein/well) were loaded and electrophoresis was carried out at constant 

 voltage (200 V) in a buffer (pH 8.3) containing 25 mM Tris-HCl and 0.19 M 

 glycine for 35 min. 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 Assay 



PRO activities were measured by adding 60 /xL PPO to 840 nl 10 mM DL- 

 DOPA in 0.05 M sodium phosphate buffer (pH 6.5) and monitoring at 25°C for 

 5 min in a Beckman DU7 spectrophotometer at 475 nm. Maximal initial 

 velocity was determined as AA^^j rn/fni" 3"^ one unit of PPO activity was 

 defined as an increase in absorbance of 0.001/min at 25°C. Unless 

 otherwise specified, experiments were carried out twice in triplicates. 

 For this study, the enzyme activities of mushroom, potato, lobster, white 

 shrimp, and brown shrimp PPO were determined to be 96,000, 120,000, 4,500, 

 1,000, and 1,200 units/mg protein, respectively. ^^ 



Anti -lobster PPO Antibody Production and Purification 



One-mL purified lobster PPO containing 100 ng protein was used as an 

 antigen to inject into a hen biweekly. Eggs laid by the immunized hen 

 were collected and anti-lobster PPO antibody was isolated and purified 

 from the egg yolk using the method of Poison et al . (1985). One part egg 

 yolk was added to 4 parts (v/v) 0.1 M sodium phosphate buffer (pH 7.6). 

 The mixture was made up 3.5% (w/v) with polyethyleneglycol (PEG) and 

 stirred for 5 min. Following centrifugation at 5,000g (10°C) for 20 min, 

 the supernatant collected was made up with 8.5% (w/v) PEG. The suspension 



• ^^■'-'. 



