.: . . '-^ ^ J-^^} . ., .:. , ■■ ,.^.. ' 19 



Each enzyme was purified further using a nondenaturing preparative 

 polyacryl amide gel electrophoresis (PAGE) system. Equipment utilized 

 included a gel tube chamber (Bio-Rad Model 175, Richmond, CA) and a Bio- 

 Rad power supply (Model EPS 500/400). A one-mL aliquot of crude enzyme 

 extract was applied to each of eight gel tubes (1.4 cm I.D. x 12 cm 

 length) containing 5% acrylamide/ 0.13% bisacrylamide gel prepared 

 according to the method of Sigma Bulletin No. MKR-137 (Sigma Chemical Co., 

 1984), and ran at a constant current of 10 mA/tube in a buffer (pH 8.3) 

 containing 5 mM Tris-HCl and 38 mM glycine. PPO was visualized using a 

 specific enzyme-substrate staining method (Constantinides and Bedford, 

 1967); 10 mM DL-^-3,4-dihydroxyphenylalanine (DL-DOPA) in 0.05 M sodium 

 phosphate buffer (pH 6.5) was used as a substrate. One tube was used to 

 determine the migration of the enzyme relative to the dye front (R^). The 

 remaining gels were then sectioned at the determined R^ and the enzyme was 

 eluted from the gel by homogenization in 0.05 M sodium phosphate buffer 

 (pH 6.5) utilizing a Bounce manual tissue grinder (Wheaton, Millville, 

 NJ). The homogenates were filtered through Whatman No. 4 filter papers, 

 pooled, and concentrated using an Amicon stirred cell (Model 8050, Amicon 

 Co., Danvers, MA) fitted with a 10 K filter (Pharmacia LBK Biotechnology 

 Inc., Piscataway, NJ). 



Extraction and Purification of Potato PPO 



The method of Patil and Zucker (1965) with some modifications was 

 used. After ammonium sulfate precipitation and dialysis, crude PPO 

 preparation was subjected to chromatography with a DEAE-cellulose (0.95 

 meq/g, Sigma) column (40 cm length x 26 mm i.d., K 26/40 Pharmacia Fine 



