152 

 and nontreated PPO were loaded individually onto the sample wells. 

 Electrophoresis was carried out at a constant voltage of 200 V for 35 min. 

 Following electrophoresis, the gel was stained with the silver stain kit 

 (Bio-Rad). The SDS-6H standard (Sigma) containing carbonic anhydrase (29 

 kD), egg albumin (45 kD) , bovine albumin (66 kD) , phosphorylase B (97.4 

 kD), j8-galactosidase (116 kD), and myosin (205 kD) was run together with 

 the samples for protein molecular weight determination. 



Pol vacrvl amide Gel Isoelectric Focusing of CO -; -treated PPO 



A gel mixture containing 4% acrylamide, 2.5% Triton X-100, 8 M urea, 

 and 5.5% ampholyte (Pharmalyte 3-10, Pharmacia) was degassed for 5 min. 

 After the addition of 5% (v/v) fresh ammonium persulfate and 0.1% (v/v) 

 N,N,N',N'-tetramethylethylenediamine (TEMED), the gel mixture was poured 

 into a 16 X 20 cm slab gel plates assembled with a 0.75 mm comb and 

 allowed to polymerize for 1.5 hr according to the Protean II Slab Cell 

 Instruction Manual (Bio-Rad Labs., 1985a). Following the removal of the 

 comb, buffer containing 0,2% (v/v) Pharmalyte 3-10 and 5% (v/v) Triton X- 

 100 was overlayed onto the polymerized gels and allowed to sit for 1 hr. 

 Prefocusings at constant voltages of 200 V (15 min), 300 V (30 min), and 

 400 V (30 min) were alternately carried out after the overlaying buffer 

 was changed (An et al . , 1989). A 50 /xL of CO2 (1 or 58 atm) -treated PPO 

 was then loaded into the sample well and electrofocusing was performed at 

 a constant voltage of 400 V for 17 hr (An et al . , 1989). The gel was 

 fixed with the fixative solution (sulfosal icylic acid/ trichloroacetic 

 acid/ methanol, 4:12.5:30, v/v) and then stained with Coomassie blue R- 

 250. The isoelectric point (pi) of PPO was determined by comparing the R^ 



