23 

 Protein Quantitat ion and Fnzvme Purity Determi nation 



The protein contents of the various PPO preparations were 

 quantitated using the Bio-Rad Protein Assay kit with bovine serum albumin 

 (Sigma) as standard. Enzyme purity was examined using a mini gel system 

 (Mini-Protean II Dual Slab Cell) (Bio-Rad, 1985b). Plant and crustacean 

 PPO's (20 ^g 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. The purity of enzyme 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 Commassie blue R-250 (Eastman 

 Kodak Co., Rochester, NY) solution. 



PPO Activity Determination 



Lobster PPO activity was determined spectrophotometrically by 

 monitoring at 475 nm the rate of dopachrome formation from DL-DOPA 

 (Savagaon and Sreenivasan, 1978). The assay was run at 25''C for 10 min by 

 mixing 40 /xL of enzyme extract with 560 iil of 10 mM DL-DOPA in 0.05 M 

 sodium phosphate buffer (pH 6.5). Two molecules of DOPA produce one 

 molecule of dopaquinone which has a molar absorption coefficient (a^,,) of 

 3,600 M-^m"^ (Fling et al . , 1963). PPO activity was defined as Atmoles 

 dopachrome formed per min at 25°C. 



PPO activities of shrimp, potato, and apple were measured by adding 

 60 Hi enzyme preparations to 840 nl 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 AA^^s ^min and one unit of PPO activity was 

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



