

151 



. Nondenaturinq Pol vacrvl amide Gel Electrophoresis of CO ^ (1 atm)-treated PPO 



Mini polyacryl amide gel with a dimension of 7 x 8 cm (1 mm 

 thickness) and containing 7.5% acrylamide/ 0.2% bisacrylamide was prepared 

 according to the Mini-Protean II Dual Slab Cell Instruction Manual (Bio- 

 Rad, 1985b). After 50 lil PPO was loaded onto the sample well, the 

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

 '^. Following electrophoresis, the gel was stained with 0.1% Coomassie blue 

 R-250 in a fixative solution (40% MeOH and 10% HOAc, v/v) for 0.5 hr and 

 then destained. The molecular weights of PPO were determined by comparing 

 the R^ values of protein with those of nondenaturing protein molecular 

 weight standards (Sigma) containing a-lactalbumin (14 kD), bovine 

 erythrocytes carbonic anhydrase (29 kD), chicken egg albumin (45 kD), 

 bovine serum albumin (monomer, 66 kD; dimer, 132 kD), and jack bean urease 

 (dimer, 240 kD; tetramer, 480 kD). 



Mass Balance of High Pressure CO -^ -treated and Nontreated PPO ' ;. 



The high pressure COj-treated PPO solution (1.5 mL) was centrifuged 

 in an Eppendorf 5415 microcentrifuge (Brinkmann Instruments Inc., Hamburg, 

 Germany) at 13,000 rpm for 30 min. After the supernatant was collected, 

 ^^>,; the pellet was redissolved in 0.5 mL 0.05 M sodium phosphate buffer (pH 

 6.5), and the protein contents of both portions quantitated using the Bio- 

 Rad Protein Assay kit. The combined protein contents of both portions 

 were then compared to that of an equal volume (1.5 mL) of nontreated PPO. 



The protein patterns of high pressure COj-treated and untreated PPO 

 were also analyzed using mini sodium dodecyl sulfate (SDS) polyacryl amide 

 gel. Fifty-/iL supernatant and pellet portions of high pressure COj-treated 



'M 



.■; )., 





