25 

 equation by measuring the initial rate of reaction at different 

 temperatures and plotting the logarithmic value of V^^ versus 1/T (Segel , 

 1976). 



To determine the thermostability, a 40 /xL aliquot of lobster PPO was 

 sealed in a quartz cell and incubated in a DU-7 spectrophotometer for 30 

 min at different temperatures ranging from 20-60°C. Following 

 equilibration to room temperature, the enzyme extract was mixed with 560 

 III of 10 mM DL-DOPA and then monitored spectrophotometrically as described 

 above. 



Molecular Weight Determination of Lobster PPO 



Sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE) 

 was used for molecular weight determination of the enzyme isoforms. Slab 

 gels (16 cm x 20 cm) at 1.50 mm thickness, consisting of stacking gel (4% 

 acrylamide/ 0.1% bisacryl amide) and separating gel (7.5% acrylamide/ 0.2% 

 bisacryl amide), were prepared according to the Protean^" II Slab Cell 

 Instruction Manual (Bio-Rad Labs., 1985a). Electrophoresis was carried 

 out in a Protean II Slab Cell system equipped with a Bio-Rad Model 

 3000/300 power supply. A constant current of 13 mA/gel and 18 mA/gel was 

 applied to the stacking and separating gel, respectively in a buffer (pH 

 8.3) containing 5 mM Tris-HCl and 38 mM glycine. Enzyme samples were 

 diluted with 4 volumes of SDS reducing buffer and then heated at 95°C for 

 4 min. Fifty-/ig aliquots were applied to each sample well. An SDS-6H 

 Molecular Weight Marker Kit (Sigma) containing carbonic anhydrase 

 (29,000), egg albumin (45,000), bovine albumin (66,000), phosphorylase B 

 (97,000), ^-galactosidase (116,000), and myosin (205,000) was used. The 



